Influence of Fluid Dynamics on Silver Nanoparticle Behavior
Total Page：16
File Type：pdf, Size：1020Kb
INFLUENCE OF FLUID DYNAMICS ON SILVER NANOPARTICLE BEHAVIOR AND MONOCYTIC CELLULAR RESPONSE Thesis Submitted to The School of Engineering of the UNIVERSITY OF DAYTON In Partial Fulfillment of the Requirements for The Degree of Master of Science in Bioengineering by Katherine Eileen Burns UNIVERSITY OF DAYTON Dayton, Ohio August 2018 INFLUENCE OF FLUID DYNAMICS ON SILVER NANOPARTICLE BEHAVIOR AND MONOCYTIC CELLULAR RESPONSE Name: Burns, Katherine Eileen APPROVED BY: _________________________________ _________________________________ Kristen K. Comfort, Ph.D. Robert J. Wilkens, Ph.D., P.E. Advisory Committee Chair Committee Member Assistant Professor Professor Chemical and Materials Engineering Chemical and Materials Engineering _________________________________ Matthew E. Lopper, Ph.D. Committee Member Associate Professor Chemistry _________________________________ _________________________________ Robert J. Wilkens, Ph.D., P.E. Eddy M. Rojas, Ph.D., M.A., P.E. Associate Dean for Research and Dean Innovation School of Engineering Professor School of Engineering ii ABSTRACT INFLUENCE OF FLUID DYNAMICS ON SILVER NANOPARTICLE BEHAVIOR AND MONOCYTIC CELLULAR RESPONSE Name: Burns, Katherine Eileen University of Dayton Advisor: Dr. Kristen K. Comfort Nanoparticles (NPs) are being increasingly used in many industries and consumer products. As they become more prevalent in consumer goods and applications, a new area of study, nanotoxicology, which explores the safety of these novel materials, has emerged. The toxicity of a particular NP can be due to many tunable physicochemical properties, such as size, core composition, morphology, and surface charge. That toxicity can also be affected by the environment surrounding the NP, including whether the system is static or dynamic, if the cells are grown within a 2-dimensional or 3- dimensional space, and the composition of the surrounding fluid. Currently, most nanotoxicity testing occurs in a standard cell-based in vitro model. These models do not take the true physiological environment in which NP exposure occurs, such as pH or the dynamic nature of the human body, into account. This investigation sought to understand some of the effects, toxicological or otherwise, of silver nanoparticles (AgNPs) on the U937 monocytic cell within both a static and dynamic exposure condition. Dynamic flow was created using a peristaltic pump, iii operating at a flow rate to produce an average tube-side linear velocity of 0.2 cm/s; the known velocity within capillaries. As the U937 cell line grew in suspension, the cells themselves were moving with the AgNPs throughout the duration of the exposure under dynamic conditions. The addition of the fluid dynamics had minimal effect on the physicochemical properties of the AgNPs themselves. However, the interactions of the AgNPs with the cells were greatly increased with the addition of the dynamic fluid movement. This increase in nano-cellular interactions also augmented AgNP-dependent bioresponses, including reactive oxygen species (ROS) production, lactate dehydrogenase (LDH) leakage, heat shock protein 27 (HSP27) activation, and activation of an inflammatory response. These observed alterations to cellular viability, stress, and inflammatory markers between static and dynamic exposure conditions suggest that the incorporation of physiologically relevant conditions in an in vitro model enhance the cellular model and could provide a mechanism to bridge the gap between in vitro and in vivo models. iv DEDICATION Dedicated to my wonderful parents, Dave and Diane, who have supported me and encouraged me to always continue learning, and to Andrew, my greatest cheerleader. v ACKNOWLEDGMENTS First, I would like to thank Dr. Kristen Comfort for her incredible guidance, support, and patience. I have learned so much about this massively broad field from her, and I am eternally grateful for the opportunity to learn from a woman as passionate about her chosen profession as she is. A special thanks to my committee as well, for taking the time out of their busy schedules to be committee members. I would also like to thank the rest of the faculty and staff of the Chemical and Materials Engineering Department at the University of Dayton. I have been supported by each and every person in the department for the last seven years, and simply would not be where I am without them. Funding for this research came from the Dayton Area Graduate Studies Institute at Wright-Patterson Air Force Base, and the National Science Foundation, each of which I am very grateful to. Finally, I owe so many thanks to my friends and family. They have pushed me to grow both academically and personally and helped to shape me into the person I am today. They are my sounding boards, confidants, and greatest cheerleaders. I am so lucky to know each and every one of them. vi TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iii DEDICATION .................................................................................................................... v ACKNOWLEDGMENTS ................................................................................................. vi LIST OF FIGURES ............................................................................................................ x LIST OF TABLES ............................................................................................................ xii LIST OF ABBREVIATIONS AND NOTATIONS ........................................................ xiii CHAPTER 1 BACKGROUND .......................................................................................... 1 1.1– Nanoparticles ........................................................................................................... 1 1.1.1 – Unique Properties of Nanoparticles ................................................................. 1 1.1.2 – Applications of Nanoparticles ......................................................................... 6 1.2 – Silver Nanoparticles ............................................................................................... 8 1.2.1 – Applications of Silver Nanoparticles ............................................................... 8 1.3 – Nanotoxicity ........................................................................................................... 9 1.3.1 – Tunable Properties and Toxicity .................................................................... 10 1.3.2 – Observable Signs of Nanotoxicity ................................................................. 13 1.4 – Physiologically Relevant Models ......................................................................... 15 vii 1.5 – References ............................................................................................................ 20 CHAPTER 2 EXPERIMENTAL PROCEDURES ........................................................... 29 2.1 – Introduction .......................................................................................................... 29 2.2 – In Vitro Cell Growth and Monitoring .................................................................. 29 2.2.1 – Cell Culture.................................................................................................... 30 2.2.2 – Cell Counting ................................................................................................. 31 2.2.3 – Cell Spinning ................................................................................................. 32 2.2.4 – Cell Lysing .................................................................................................... 33 2.3 – NP Characterization ............................................................................................. 34 2.3.1 – Transmission Electron Microscopy (TEM) ................................................... 35 2.3.2 – Dynamic Light Scattering (DLS) .................................................................. 36 2.3.3 – Zeta Potential ................................................................................................. 37 2.3.4 – Ultraviolet-Visible Spectroscopy (UV-Vis) .................................................. 38 2.4 – NP Exposure and Response Characterization ...................................................... 39 2.4.1 – AgNP Exposures............................................................................................ 39 2.4.2 – AgNP Internalization by U937 Cells ............................................................. 44 2.5 – Cellular Responses to NP Exposure ..................................................................... 46 2.5.1 – Lactate Dehydrogenase (LDH) ...................................................................... 47 2.5.2 – Reactive Oxygen Species (ROS) ................................................................... 48 2.5.3 – Cytokine ELISAs ........................................................................................... 53 viii 2.6 – Signaling Responses to NP Exposure .................................................................. 55 2.6.1 – BCA Protein Assay (BioRad) ........................................................................ 55 2.6.2 – Stress ELISAs ................................................................................................ 57 2.7 – Statistical Analysis ............................................................................................... 59 2.8 – References