Nitrogen Oxide Delivery Systems for Biological Media by Brian Thomas Skinn B.S. Chemical Engineering (2004) Case Western Reserve University Submitted to the Department of Chemical Engineering In Partial Fulfillment of the Requirements of the Degree of DOCTOR OF PHILOSOPHY IN CHEMICAL ENGINEERING at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY February 2012 © Massachusetts Institute of Technology 2012 All Rights Reserved Signature of Author .............................................................................................................. Department of Chemical Engineering December 2, 2011 Certified by .......................................................................................................................... William M. Deen Carbon P. Dubbs Professor of Chemical Engineering Thesis Supervisor Accepted by ......................................................................................................................... William M. Deen Carbon P. Dubbs Professor of Chemical Engineering Chairman, Committee for Graduate Students Nitrogen Oxide Delivery Systems for Biological Media by Brian Thomas Skinn Submitted to the Department of Chemical Engineering on December 2, 2011 in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Chemical Engineering ABSTRACT Elevated levels of nitric oxide (NO) in vivo are associated with a variety of cellular modifications thought to be mutagenic or carcinogenic. These processes are likely mediated by reactive nitrogen species (RNS) such as nitrogen dioxide (NO2) and peroxynitrite formed from the respective reactions of NO with oxygen and superoxide anion. Controlled delivery of these RNS at levels expected to occur in vivo is desirable in studying these processes and their role in the etiology of various diseases. Two delivery systems were developed that provide novel capabilities for steady, quantitative exposure of biological targets to RNS over periods from hours to days. Quantitative models are presented that accurately describe the behavior of both systems. The first system achieves NO concentrations of 0.6-3.0 μM in a stirred, liquid-filled vessel by diffusion from a gas stream through a porous poly(tetrafluoroethylene) membrane. Oxygen, consumed by reaction with NO or by other processes, is supplied by diffusion from a separate gas stream through a loop of poly(dimethylsiloxane) tubing. The adventitious chemistry observed in a prior device for NO delivery [Wang C. Ann Biomed Eng (2003) 31:65-79] is eliminated in the present design, as evidenced by the close match to model predictions of the accumulation rate of nitrite, the stable end product of NO oxidation. The second system delivers NO2 by direct contacting of a stirred liquid with an NO2- containing gas mixture. Accumulation rates of products in the presence and absence of the NO2-reactive substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) matched model predictions within 15% for all conditions studied. The predicted steady NO2 concentration in the liquid is on the order of 400 pM, similar to what is expected to be present in extracellular fluids in the presence of 1 μM NO. This system appears to be the first reported with the capability for sustained, quantitative NO2 delivery to suspended cell cultures. Results from initial efforts to test a novel mixing model for bolus delivery of peroxynitrite to agitated solutions imply that the proposed model might accurately describe mixing in bolus delivery experiments with agitation by vortex mixing, but further work is required to validate the model. Thesis Supervisor: William M. Deen Title: Professor of Chemical Engineering 3 Acknowledgements This thesis would not have been possible without the assistance and encouragement of a number of people. Foremost among these is my advisor, Prof. William Deen, who has been an inexhaustible and invaluable source of guidance and input as I wrestled with the chemistry of nitrogen oxides. I am especially grateful for his assistance in improving the concision, precision, and clarity of my technical communication. I am also indebted to the members of my thesis committee, Professors Klavs F. Jensen (Chemical Engineering) and Steven R. Tannenbaum (Biological Engineering), whose comments, suggestions, and criticisms were invaluable in guiding my research. Thanks also to the members of Deen group, including Kristin Mattern, Gaurav Bhalla, Chang Hoon Lim, and Melanie Chin, both for engaging and helpful technical discussions and for making the lab environment fun and easygoing. I greatly appreciate the experimental assistance provided by UROPs George Pratt and Deborah Markham. Debbie’s patience and unflappable good cheer in the face of a long series of generally unsuccessful experiments were truly exemplary. I am also grateful for the technical assistance rendered by Mark Belanger (Edgerton student machine shop, MIT) in my efforts to fabricate the custom NO reactor components. I am indebted to Laura Trudel, Pete Wishnok, Yu Zeng, Vicki Dydek and Nick Zaborenko for their assistance with lines of experimental inquiry that were, in the end, unfruitful. The social environment of the department was thoroughly enjoyable. Props go to Arman Haidari, Wyatt Tenhaeff and Michael Zahniser for enduring my idiosyncrasies during the Upland years. I also am grateful to Kristin, Jason, Sanjoy, Nick, Heather, and company for introducing me to the finer details of the sugar, coffee, and corn markets, and to Dave Adrian and Micah Green for the in-depth tours of the fast-paced and frequently explosive world of sprockets. I would be remiss if I failed to acknowledge the innumerable ways in which my parents have assisted me throughout the years. They have provided much appreciated assistance in matters large and small throughout all of my (many!) years of education. Finally, this thesis would not have been possible without the constant encouragement and unfailing support of my wife Lisa. She is always ready to celebrate any success, large or small, and was always there with a word of encouragement in those long days of, “Well, the data is good, but I have no idea what it means.” I look forward to sharing with her all that awaits us in the years to come. 5 Table of Contents Chapter 1 Introduction and Background ......................................................................... 17 1.1 Nitric oxide in biology ..................................................................................... 17 1.2 Biological chemistry of nitrogen oxides .......................................................... 20 1.3 Delivery of nitrogen oxides.............................................................................. 24 1.3.1 Reported methods................................................................................ 24 1.3.2 Overview and evaluation...................................................................... 29 1.4 Research objectives.......................................................................................... 33 Chapter 2 Nitric Oxide Delivery System for Biological Media ...................................... 35 2.1 Introduction...................................................................................................... 35 2.2 Materials and methods ..................................................................................... 38 2.2.1 Chemicals............................................................................................. 38 2.2.2 Gas delivery system ............................................................................. 38 2.2.3 Apparatus ............................................................................................. 39 2.2.4 Concentration measurements ............................................................... 41 2.2.5 NO oxidation kinetics .......................................................................... 41 2.2.5.1 Substrate-free media ............................................................... 41 2.2.5.2 NO oxidation of morpholine................................................... 42 2.2.6 Reactor model ...................................................................................... 43 2.2.6.1 Macroscopic model................................................................. 43 2.2.6.2 Derivation of the correction factors χj and γ........................... 44 2.2.7 Evaluation of mass transfer coefficients .............................................. 47 2.2.7.1 Mass transfer coefficients for O2 ............................................ 47 2.2.7.2 Mass transfer coefficients for NO........................................... 48 2.2.8 Tests using nitrite and NMor accumulation......................................... 49 2.2.9 Simultaneous delivery of NO and O2................................................... 51 2.3 Results.............................................................................................................. 52 2.3.1 Mass transfer coefficients .................................................................... 52 2.3.2 Tests using nitrite and NMor accumulation......................................... 52 2.3.3 Simultaneous delivery of NO and O2................................................... 60 7 2.4 Discussion ........................................................................................................ 64 2.4.1 Elimination of adventitious NO oxidation ........................................... 64 2.4.2 Model performance .............................................................................