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Characterization and Functionalization of Iron-Oxide Nanoparticles for Use As Potential Agents for Cancer Thermotherapy at the U

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Characterization and Functionalization of Iron-Oxide Nanoparticles for Use As Potential Agents for Cancer Thermotherapy at the U Characterization and Functionalization of Iron-Oxide Nanoparticles for Use as Potential Agents for Cancer Thermotherapy By Nora O’Reilly A dissertation submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy (Animal Science) At the University of Wisconsin-Madison 2013 Date of final oral examination: April 18th, 2013 The dissertation is approved by the following members of the Final Oral Committee: Professor Ralph Albrecht, Animal Science, Pediatrics, Pharmaceutical Sciences Professor Mark Cook, Animal Science Professor Amin Fadl, Animal Science Professor Manish Patankar, Obstetrics and Gynecology Professor Chris Brace, Radiology, Biomedical Engineering i Abstract This thesis presents experimental studies of iron oxide nanoparticle synthesis, functionalization, and intracellular hyperthermal effects on murine macrophages as a model in vitro system. Colloidal suspensions of magnetic nanoparticles (MNPs) are of particular interest in Magnetic Fluid Hyperthermia (MFH). Iron oxide nanoparticles (IONPs) have garnered great interest as economical, biocompatible hyperthermia agents due to their superparamagnetic activity. Here we seek to optimize the synthetic reproducibility and in vitro utilization of IONPs for application in MFH. We compared aqueous synthetic protocols and various protective coating techniques using various analytical techniques and in vitro assays to assess the biocompatibility and feasibility of the various preparations of nanoparticles. Using a co-precipitation of iron salts methodology, iron oxide nanoparticles (IONPs) with an average diameter of 6-8nm were synthesized and stabilized with carboxylates. By performing calorimetry measurements in an oscillating magnetic field (OMF) with a frequency of 500 kHz and field strength of 0.008Tesla the superparamagnetic behavior of these particles was confirmed. To further investigate these IONPs in a biological application, citric acid-stabilized particles, in conjunction with heat generated by these IONPs when exposed to an OMF, were assessed to determine their effects on cell viability in a RAW 267.4 murine macrophage model system. Our results show that 91.5-97% of cells that have ingested IONPs die follow exposure to an OMF. Importantly, neither the IONPs (at applicable concentrations) nor the OMF show cytotoxic effects. These particular particles have promising preliminary results as hyperthermic agents in both the current literature and simple, proof-of-concept experiments in our laboratory setting. We present experimental results for the synthesis, characterization, and utilization of iron ii oxide nanoparticles in MFH. Our results show that while IONPs have potential in MFH, efforts to advance IONPs from laboratory to clinical contexts, where reliable generative techniques and consistent performance properties are necessary, will require an understanding of the influence of the diverse intrinsic structural and magnetic characteristics as well as surface chemistry of nanoparticles, as well as the mechanisms of particle uptake and cell death due to intracellular hyperthermia. iii Acknowledgements There are an enormous number of people who have contributed not only to this dissertation, but my development and evolution as a student, teacher, and a scientist. Of these people who have been enormously supportive in the preparation of this thesis, I am especially thankful to my advisor, Dr. Ralph Albrecht, for all of his efforts in guiding me through to its successful completion. Dr. Albrecht has been an exceptional mentor, from the time he introduced to me to research as the professor in my undergraduate senior seminar in UW-Madison’s Animal Science department, through my years as a graduate student in his group in the same department. He always made himself available to provide guidance on the numerous challenges that I encountered during my studies and research. He has taught me invaluable skills as a scientific researcher and critic, especially the ability to approach scientific questions with a solid understanding of cellular biology, immunology, and nanotechnology. Again, I express my most sincere gratitude to Ralph for his support in my development as a scholarly researcher. My special thanks to the members of my advisory committee: Professor Mark Cook, Professor Amin Fadl, Professor Manish Patankar, and Professor Chris Brace for their valuable time, suggestions, and discussions. My heartfelt thanks to Dr. Ian Rowland for all his valuable work with our small animal MRI proof-of-concept feasibility studies. I am sorry that circumstances did not allow for us to continue our work together in greater depth. Ian’s enthusiasm, compassion, and sense of humor were equally appreciated as the expertise in his field that he shared so freely. iv Someone who quietly carriers the lab on his shoulders is our lab manager, Joseph Heintz. Professor Heintz taught me all the laboratory and analytical techniques that were utilized in this dissertation. As Joe’s skill as an electron microscopist far exceeds my own novice abilities, he kindly performed much of the imaging that is included in the figures for this work. I truly would have been an elephant in a china shop had it not been for Joe’s supervision, guidance, and advice. I would also like to thank the many collaborators and lab members for their advice and suggestions throughout the years including Professor Julie Oliver, Professor Doug Steeber, Professor Marija Gajdardziska-Josifovska, Evan Krystofiak, and Eric Mattson from the University of Wisconsin-Milwaukee. A post-doctorate researcher under Professor Song Jin in the Chemistry department, Chad Dooley, was immensely helpful in his explanations of the particle and surface chemistry involved in the synthesis and stabilization of our particles. His passion for chemistry was inspiring, carrying me some of the most frustrating periods of this research. While there are many undergraduate students with whom I had the pleasure of working, the current students in the Albrecht lab who have been so helpful during the completion of this dissertation are Diana Perdomo and Kaitlyn Soukup. I am also thankful for Tom Tabone, Animal Science’s resident statistician, and his ability to be bribed with baked goods. Tom was instrumental in the statistical analysis included in this dissertation. v At some point these dedications begin to take on an Oscar-night quality, so I must wrap up with thanks to all the professors from the UW Department of Neuroscience who taught me that the material professors impart is secondary to the inspiration and life they breathe into their material, igniting the minds and souls of future generations, the Delta program for providing formal training regarding teaching at the university level, and Liv Sandberg who was kind enough to allow me to flex my newly discovered teaching muscles in her classroom. Lastly, but always first in my heart, thank you to my family and friends for listening to my excited, if slightly frenetic explanations of what I am actually doing in school and supporting me in all my academic endeavors in countless ways, both large and small. It would have been impossible for me to successfully complete my graduate education had it not been for your love. vi Table of Contents Characterization and Functionalization of Iron-Oxide Nanoparticles for Use as Potential Agents for Cancer Thermotherapy .............................................................................................. i Abstract ........................................................................................................................................... i Acknowledgements ...................................................................................................................... iii Chapter 1 ....................................................................................................................................... 1 Introduction ............................................................................................................................... 1 Introduction to Magnetic Nanoparticles ......................................................................... 1 Biological Applications of Magnetic Nanoparticles ....................................................... 2 Magnetic Domains ............................................................................................................ 3 Inductive Heating of Magnetic Nanoparticles ................................................................ 4 Biomedical Application of the Magnetic Properties of Nanoparticles ......................... 4 Diagnostic In Vivo Imaging.............................................................................................. 5 Therapeutics ...................................................................................................................... 5 Introduction to Tumor Hyperthermia ............................................................................ 6 Harnessing the Potential of Superparamagnetism and the Inductive Heating Phenomena......................................................................................................................... 6 Nanotheranostics ............................................................................................................... 7 Background and Motivation ............................................................................................ 8 Scope of Thesis .................................................................................................................
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