Synthesis and Biophysical Characterization of Polymerized Hemoglobin Dispersions of Varying Size and Oxygen Affinity as Potential Oxygen Carriers for use in Transfusion Medicine Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yipin Zhou, M.S., B.S. Graduate Program in Chemical and Biomolecular Engineering The Ohio State University 2011 Dissertation Committee: Andre Francis Palmer, Advisor Jeffrey J. Chalmers Shang-Tian Yang Copyright by Yipin Zhou 2011 ABSTRACT Blood transfusion can be compromised by a number of physiological and practical issues such as the risk of contracting infectious diseases, initiation of harmful immunological responses, the red blood cell (RBC) storage lesion and the shrinking availability of RBCs. Thus, there is a need to develop safe and efficacious O2 carriers for use in transfusion medicine as RBC substitutes in order to maintain proper tissue and organ oxygenation. Hemoglobin (Hb) is the most prevalent protein inside the RBC and is the natural carrier of O2 in vivo. Therefore, Hb-based O2 carriers (HBOCs) are considered as good candidates for RBC substitutes. Currently, HBOCs can be manufactured by conjugation of molecules to the surface of Hb, encapsulation of Hb inside particles, site-directed mutagenesis of Hb and cross- linking/polymerizing Hb. Among these approaches, polymerization of human or bovine Hb with the difunctional cross-linking reagent glutaraldehyde represents a simple strategy to synthesize HBOCs. In fact, the two commercial polymerized Hb (PolyHb) products Hemopure® (glutaraldehyde polymerized bovine Hb, OPK Biotech, Cambridge, MA) and PolyHeme® (pyridoxalated glutaraldehyde polymerized human Hb, Northfield Laboratories Inc., Evanston, IL), which have failed Phase III clinical trials, are based on this approach. These commercial PolyHb solutions face serious safety issues including ii the induction of vasoconstriction in the microcirculation and the development of systemic hypertension. These side-effects are due to the existence of the Hb tetramer or dimer in the blood, which subsequently extravasate through the blood vessel wall and scavenge the vasodilator nitric oxide (NO) or trigger an autoregulatory response of the blood vessel to reduce the oversupply of O2 to surrounding tissues. Therefore, the goal of this research is to synthesize a new generation of HBOCs with fewer side-effects, longer circulation lifetime in the blood and better oxygenation potential. In this research, we hypothesize that increasing HBOC size will reduce vasoconstriction in the microcirculation, systemic hypertension as well as oxidative damage to tissues and organs. We propose to synthesize a small library of PolyHbs of varying size by cross-linking/polymerizing bovine Hb with the cross-linking agent glutaraldehyde. Also, since there is a lot of debate in the blood substitute research community about the effect of O2 affinity on vasoactivity and hypertension, we will engineer PolyHb O2 affinity by synthesizing PolyHb with both low oxygen affinity (L- PolyHb) and high oxygen affinity (H-PolyHb). After synthesizing the PolyHb solutions, two mathematical models will be developed with the finite element analysis software COMSOL Multiphysics (COMSOL, Burlington, MA) to evaluate the ability of PolyHbs to transport O2 both in a hepatic hollow fiber bioreactor and an arteriole. In this dissertation, we demonstrated that by maintaining bovine Hb (bHb) in either the low O2 affinity tense state (T-state) or high O2 affinity relaxed state (R-state) during the polymerization reaction and purifying the PolyHb via tangential flow filtration, we were able to synthesize novel ultrahigh molecular weight (MW) PolyHbs with distinct O2 affinities with no tetrameric Hb, high viscosity, low colloid osmotic pressure and the iii ability to maintain O2 dissociation, CO association and NO dioxygenation reactions. The PolyHbs caused less in vitro RBC aggregation than 6% dextran (500 kDa) and underwent little dissociation in vivo. Then, we systematically investigated the effect of varying the glutaraldehyde to Hb (G:Hb) molar ratio on the biophysical properties of PolyHb polymerized in either the low or high O2 affinity state. Our results showed that the MW and molecular diameters of the resulting PolyHbs increased with increasing G:Hb molar ratio. For low O2 affinity PolyHbs, increasing the G:Hb molar ratio reduced the O2 affinity while for high O2 affinity PolyHbs, increasing the G:Hb molar ratio led to increased O2 affinity compared to unmodified bHb and low O2 affinity PolyHbs. In addition, increasing the G:Hb molar ratio could increase the zeta (ζ) potential of L-PolyHbs making them more stable in aqueous solution. However, both L- and H-PolyHbs had higher autoxidation rates than unmodified bHb with L-PolyHbs autoxidizing faster than H-PolyHbs. All PolyHbs displayed higher viscosities compared to unmodified bHb and whole blood, which also increased with increasing G:Hb molar ratio. In contrast, the colloid osmotic pressure of PolyHbs decreased with increasing G:Hb molar ratio. Two mathematical models were developed after investigating the synthesis and biophysical properties of the PolyHbs. In an O2 transport model of a hepatic hollow fiber bioreactor, L-PolyHbs showed similar oxygenation ability to the commercial product Oxyglobin® (glutaraldehyde polymerized bovine Hb, OPK Biotech, Cambridge, MA) and oxygenated the bioreactor better than H-PolyHbs. In a combined NO and O2 transport model in an arteriole facilitated by PolyHb solutions, high viscosity PolyHb solutions promoted blood vessel wall shear stress dependent generation of the vasodilator NO iv especially in the vicinity of the blood vessel wall compared to the commercial PolyHb Oxyglobin® although NO scavenging in the arteriole lumen was unavoidable. We also observed that all PolyHbs could improve tissue oxygenation under anemic conditions under hypoxic conditions, while L-PolyHbs were more effective under normoxic conditions than H-PolyHbs. In addition, all ultrahigh MW PolyHb displayed higher O2 transfer rates than the commercial HBOC Oxyglobin®. This project is significant in that it is a systematic investigation of the synthesis, biophysical properties and theoretical oxygenation abilities of PolyHb polymerized with either low (L) or high (H-) oxygen affinity. The knowledge gained from this study should guide the design of the next generation of PolyHbs for use in tissue engineering and transfusion medicine. v Dedicated to my wife and parents vi Acknowledgements I am heartily thankful to my advisor, Professor Andre F. Palmer, whose encouragement, guidance and support from the initial to the final level enabled me to develop a thorough understanding of the subject throughout my research. This dissertation would not have been possible without his invaluable guidance and support. I would like to thank my fellow group members. I would like to thank Dr Guoyong Sun from whom I learnt the techniques of hemoglobin polymerization and characterization. I would like to thank Dr Xuesong Jiang for his instruction in the mammalian cell culture and operation of bioreactor. My gratitudes also go to Dr Guo Chen and Dr Gundersen who gave me constructive advice in simulation. I would also like to thank my lab-mates Ning Zhang, Jacob J. Elmer, David Harris and Shahid Rameez for their supports and encouragements. My thanks are extended to Dr Buehler at FDA and Dr Cabrales at UCSD for their supports and guidances for this research. I would also like to take this opportunity to specially thank my wife Li Ling. Without her support, I would have achieved nothing. vii Vita Febrary 1982 …………………………………………………Born-Shanghai, PR China June 2004 …………………………………………………………… B.S. Bioengineering East China University of Science and Technology, Shanghai, PR China June 2007 …………………………………………………………… M.S. Microbiology East China University of Science and Technology, Shanghai, PR China Publications 1. Buehler PW, Zhou Y, Cabrales P, Jia Y, Sun G, Harris DR, Tsai AG, Intaglietta M, Palmer AF. Synthesis, biophysical properties and pharmacokinetics of ultrahigh molecular weight tense and relaxed state polymerized bovine hemoglobins. Biomaterials 2010;31(13):3723-3735. 2. Zhou Y, Jia Y, Buehler PW, Cabrales P, Chen G, Palmer AF. Synthesis, biophysical properties and oxygenation potential of high molecular weight polymerized bovine hemoglobins with low and high oxygen affinity. Biotechnology progress 2011;27(4):1172-1184 3. Cabrales P, Zhou Y, Harris DR, Palmer AF. Tissue oxygenation after exchange transfusion with ultrahigh-molecular-weight tense- and relaxed-state polymerized bovine hemoglobins. Am J Physiol Heart Circ Physiol 2010;298(3):H1062-1071. FIELDS OF STUDY Major Field: Chemical and Biomolecular Engineering viii TABLE OF CONTENTS ABSTRACT··························································································ii DEDICATION·····················································································vi ACKNOWLEDGEMENTS·······································································vii VITA································································································viii LIST OF TABLES········································································xiii LIST OF FIGURES················································································xiv CHAPTER 1 Introducion···········································································1