Synthesis and Biophysical Characterization of Polymerized

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

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

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.

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

1.1 Motivation························································································1

1.1.1 Risks of infectious diseases and immunological reactions in transfused RBCs·····2

1.1.2 Storage lesion of RBCs··································································3

1.1.3 Limited supply of blood·································································4

1.2 Types of artificial O2 carriers··································································4

1.2.1 PFCOCs···················································································5

1.2.2 Background on Hb·······································································7

1.2.3 HBOCs···················································································10

1.2.3.1 Stroma-free Hb as an HBOC················································10

1.2.3.2 Strategies to modify Hb······················································12

ix 1.2.4 Ultra high MW PolyHbs can address the side-effects of acellular HBOCs·····16

1.3 Objective of Dissertation······································································19

CHAPTER 2 Synthesis, Biophysical Properties and Pharmacokinetics of Ultrahigh MW

PolyHbs······························································································21

2.1 Introduction·····················································································21

2.2 Materials and Methods········································································23

2.2.1 Materials·················································································23

2.2.2 Hb Purification··········································································24

2.2.3 Polymerization of Hb··································································24

2.2.5 Desalting and Buffer Exchange of PolyHb solutions····························26

2.2.6 MetHb Level and Protein Concentration of PolyHb Solutions···················27

2.2.7 SDS-PAGE of PolyHb Solutions·····················································28

2.2.8 Size Exclusion Chromatography (SEC) Coupled with Multi-Angle Static Light

Scattering (MASLS) Analysis of PolyHb Solutions······························28

2.2.9 O2-PolyHb Equilibria··································································29

2.2.10 Stopped Flow Kinetic Analysis of PolyHb Solutions····························29

2.2.11 PolyHb Viscosity and COP··························································31

2.2.12 RBC Aggregation in PolyHb Solutions············································31

2.2.13 In Vitro Autoxidation of PolyHb Solutions········································32

2.2.14 Animals and Surgical Preparation··················································33

2.2.15 Pharmacokinetic Analysis of PolyHb Solutions··································34

2.3 Results···························································································35

x 2.3.1 pO2 of PolyHb Solutions·······························································36

2.3.2 SDS-PAGE and MW Distribution of PolyHb Solutions··························37

2.3.3 P50 and n of PolyHb Solutions························································39

2.3.4 MetHb Level of PolyHb Solutions···················································41

2.3.5 Stopped Flow Kinetic Analysis of PolyHb Solutions······························41

2.3.6 Viscosity and COP of PolyHb Solutions············································43

2.3.7 RBC Aggregation of PolyHb Solutions·············································43

2.3.8 Pharmacokinetic Analysis of PolyHb Solutions····································45

2.3.9 Plasma PolyHb Polymer Dissociation···············································48

2.3.10 Oxidation of L- and H-PolyHb······················································49

2.4 Discussion·······················································································50

2.4.1 Biophysical Properties of L- and H-PolyHb Solutions····························50

2.4.2 Pharmacokinetics of L- and H- PolyHb Solutions·································56

2.5 Conclusions·····················································································57

CHAPTER 3 The Effect of Cross-link Density on the Biophysical Properties and

Oxygenation Potential of PolyHbs with Low and High O2 Affinity·························58

3.1 Introduction·····················································································58

3.2 Materials and Methods········································································60

3.2.1 Materials·················································································60

3.2.2 Hb Purification··········································································60

3.3.3 Polymerization of bHb·································································60

3.2.4 Clarification and Diafiltration of PolyHb Solution·································60

xi 3.2.5 MetHb Level and Protein Concentration of PolyHb Solutions···················61

3.2.6 Equilibria of O2-bHb/PolyHb Solutions ············································62

3.2.7 SDS-PAGE and Native-PAGE of PolyHb Solutions······························62

3.2.8 Absolute MW Distribution of bHb/PolyHb Solutions·····························62

3.2.9 Viscosity and Colloid Osmotic Pressure of bHb/PolyHb Solutions·············62

3.2.10 Stopped Flow Kinetic Analysis of bHb/PolyHb Solutions······················63

3.2.11 Hydrodynamic Molecular Size and Zeta Potential of bHb/PolyHb············65

3.2.12 In Vitro Autoxidation of PolyHb Solutions········································65

3.2.13Simulation of bHb/PolyHb Facilitated O2 Transport in a Hepatic HF

Bioreactor··············································································66

3.3 Results···························································································69

3.3.1 pO2 of bHb Solutions During the Polymerization Process························69

3.3.2 Effect of Cross-link density on MW Distribution of PolyHb Solutions·········70

3.3.3 Effect of Cross-link Density on Hydrodynamic Diameter and Zeta Potential of

PolyHbs………………………………………………………………………72

3.3.4 Effect of Cross-link density on Oxygen Affinity and Cooperativity of PolyHb

Solutions·················································································73

3.3.5 Effect of Cross-link Density on MetHb Level of PolyHb Solutions·············75

3.3.6 Effect of Cross-link Density on Viscosity and COP of PolyHb Solutions······75

3.3.7 Effect of Cross-link Density on Stopped Flow Kinetic Analysis of PolyHb

Solutions················································································76

3.3.8 Autoxidation of PolyHb································································77

xii 3.3.9 Simulation of bHb/PolyHb Facilitated O2 Transport in a Hepatic HF

Bioreactor···············································································78

3.4 Discussion·······················································································82

3.5 Conclusions·····················································································93

CHAPTER 4 Simulation of NO and O2 Transport Facilitated by PolyHb Solutions in an

Arteriole······························································································94

4.1 Introduction·····················································································94

4.2 Computational Methods·······································································96

4.2.1 Hb-O2 Release/Binding Kinetics······················································97

4.2.2 Mass Balance on O2/NO with Hb Encapsulated within RBCs and HBOCs····99

4.2.2.1 Arteriole Lumen (0≤r≤r2) ····················································99

4.2.2.2 Glycocalyx Layer (r2

4.2.2.3 Endothelial Cell Layer (r3

4.2.2.4 Interstitial Layer (r4

4.2.2.5 Smooth Muscle Cell Layer (r5

4.2.2.6 Tissue Space (r6

4.2.3 Model Parameters·····································································104

4.3 Results: ························································································108

4.3.1 Effect of PolyHb on NO Profiles····················································108

4.3.2 pO2 Profiles Upon Transfusion of Hb/PolyHb solutions························112

4.3.3 O2 Transfer Rate of PolyHbs························································116

4.4 Discussion·····················································································119

xiii 4.5 Conclusions····················································································127

CHAPTER 5 Conclusions and Future Work··················································128

5.1 Summary and conclusions···································································128

5.2 Future work····················································································130

5.2.1 Conjugation of PolyHb with antioxidant enzymes·······························130

5.2.2 Scale-up of polymerization process·················································131

5.2.3 Carbon monoxide saturated PolyHb for use in transfusion medicine··········132

Reference: ·························································································133

xiv LIST OF TABLES

Table 2.1: Rapid kinetic parameters of gaseous ligand reactions with unmodified bHb, L- and H-PolyHb ················································································43

Table 2.2: The viscosity and COP of L- and H-PolyHb·······································43

Table 2.3: Pharmacokinetic parameter following L- and H-PolyHb transfusion··········47

Table 3.1: Biophysical properties of bHb/PolyHb solutions··································72

Table 3.2: Molecular diameter and zeta potential of bHb/PolyHb ··························73

Table 3.3: Viscosity and COP of bHb/PolyHb solutions······································76

Table 3.4: Kinetic parameters of bHb/PolyHb solutions······································77

Table 3.5: Autoxidation rate constant of bHb/PolyHb solutions·····························77

Table 4.1: Constants and parameters used in the Simulations······························104

Table 4.2: Physical properties of PolyHbs and other HBOCs·······························106

Table 4.3: Diffusivity and adair constants of PolyHbs and other HBOCs·················106

Table 4.4: Blood viscosity and blood vessel wall shear stress after transfusion······107

Table 4.5: Comparison of estimated blood vessel wall shear sress against measured Blood vessel wall shear stress·····································································120

xv LIST OF FIGURES

Figure 1.1: Comparison of the O2 carrying capacity between whole human blood and the

PFC Oxygent® [1] ··················································································6

Figure 1.2: A: The structure of human Hb (PDB 1GZX) [2] and B: The structure of the heme group [3] ······················································································7

Figure 1.3: A: The O2 equilibrium curve of Hb; B The transition of Hb between the T- state (PDB 4HHB) [4] and the R-state (PDB 1HHO)[5] ····································8

Figure 2.1: pO2 at various stages of the bHb polymerization process for L- and H-PolyHb solutions······························································································36

Figure 2.2: SDS-PAGE of unmodified bHb, L- and H-PolyHb solutions··················37

Figure 2.3: Absolute MW distribution of unmodified bHb, L- and H-PolyHb solutions······························································································38

Figure 2.4: Equilibrium O2-bHb/PolyHb binding curves of unmodified bHb, L- and H-

PolyHb solutions············································································39

Figure 2.5: Oxygen affinity (P50) and cooperativity coefficient (n) of unmodified bHb, L- and H-PolyHb solutions········································································40

Figure 2.6: MetHb level of bHb and PolyHb solutions········································41

Figure 2.7: Rapid kinetics of CO binding with unmodified deoxygenated bHb, L- and H-

PolyHb··················································································42

Figure 2.8: A typical blood smear for aggregation studies ···································44

xvi Figure 2.9: A: Pharmacokinetics of L-PolyHb; B: Pharmacokinetics of H-PolyHb·······45

Figure 2.10: Circulating PolyHb polymer distribution over time····························48

Figure 2.11: In vitro autoxidation of L- and H-PolyHg·······································49

Figure 3.1: Hepatic HF bioreactor system·······················································68

Figure 3.2: SDS-PAGE of bHb/PolyHb solutions··············································70

Figure 3.3: Native-PAGE of bHb/PolyHb solutions···········································70

Figure 3.4: Absolute MW distribution of bHb/PolyHb solutions····························71

Figure 3.5: O2-bHb/PolyHb equilibrium curves················································75

Figure 3.6: O2 consumption rate normalized by the control (no HBOC) ···················78

Figure 3.7: Steady state pO2 profiles in a single hollow fiber································80

Figure 3.8: ECS Zonation··········································································81

Figure 4.1: Model Geometry of Arteriole·······················································97

Figure 4.2: The effect of increasing blood vessel wall shear stress upon transfusion of

PolyHb solutions on the NO concentration profiles··········································109

Figure 4.3: The average NO concentration in the endothelial cell layer···················110

Figure 4.4: The relationship between blood vessel wall shear stress and average NO concentration········································································111

Figure 4.5: Steady state pO2 profiles upon transfusion of PolyHb at hct = 11%·········112

Figure 4.6: Steady state pO2 profiles upon transfusion of PolyHb at hct = 18%·········113

Figure 4.7: O2 transfer rate across the arteriole at different hcts and inlet pO2s··········118

xvii CHAPTER 1

Introduction

1.1 Motivation

Oxygen (O2) is essential for the survival of all aerobic organisms and is a key substrate in aerobic cellular respiration, which facilitates the storage of energy in the portable form of adenosine-5'-triphosphate (ATP) [6, 7]. ATP can then be used to drive energy-intensive processes, including active transport of molecules across membranes [8], generation of force and movement in cells and muscles [9, 10], cellular signal transduction [11, 12] and biosynthesis of macromolecules such as DNA [13, 14] and

RNA [15]. Therefore, facilitation of O2 transport is critical for maintaining or increasing the performance of aerobic organisms. This is typically achieved through the use of an O2 carrier that is normally present in the circulatory system of the aerobic organism.

For example, red blood cells (RBCs) are the primary carriers of O2 in the systemic circulation of vertebrates, which facilitate its transport from the lungs to the tissues and organ systems in vivo [16]. In the absence of human RBCs, the solubility of O2 in human blood plasma is very low (~ 0.3 ml/dL at 37℃ and 1 atm of air). Fortunately, human

RBCs present in human blood increase the solubility of O2 to ~ 7 ml/dL at a RBC volume fraction (i.e. hematocrit, Hct) of ~45% [17]. Therefore, it becomes evident that the increased O2 carrying capacity of human whole blood compared to plasma is essential to

1 the maintenance of human life.

In light of the O2 storage and transport functions of RBCs, allogeneic RBC transfusion has long been considered an important treatment option for patients suffering from massive blood loss [18] due to trauma and/or anemia [19] by maintaining adequate oxygenation of tissues and organs. In fact, RBCs are transfused almost every two seconds in the United States (US) [20]. However, transfusion of RBCs is not entirely risk-free and can be compromised by a number of physiological and practical problems [21].

1.1.1 Risks of infectious diseases and immunological reactions in transfused RBCs

Public concern about the safety of RBC transfusion continues to the present since the emergence of human immunodeficiency virus (HIV) infection in the 1980s’, which can be transmitted via transfusion of infected blood products [22]. Currently, the American

Red Cross tests donated blood for hepatitis B and C viruses, HIV, human T-cell lymphotropic virus, syphilis, West Nile virus and the agents of Chagas disease and variant Creutzfeldt-Jacob disease (vCJD) [23-29]. As a result, the safety of the US blood supply in terms of transfusion-transmitted diseases is quite good. However as new infectious agents emerge, the costs associated with producing a unit of RBCs increases; since additional screening tests may have to be conducted before RBCs can be distributed to health care providers. Of more concern is the fact that donated blood may contain yet to be identified infectious agents [25]. Moreover, the safety of the blood supply in developing countries is still severely threatened by the blood-borne pathogens, since limited tests are done to screen the donated blood for pathogens [30]. In addition, RBC transfusion is sometimes accompanied by adverse immunological reactions such as

2 delayed hemolysis [31], severe transfusion-related acute lung injury (TRALI) [32] and even mistransfusion [33].

1.1.2 Storage lesion of RBCs

The RBC storage lesion is another serious concern regarding the safety and efficacy of RBC transfusion following extended duration storage periods [34-36]. More specifically, transfusion of long-term stored RBCs may cause various severe adverse side-effects in patients, including increased gut ischemia [37], increased mortality after transfusion [38, 39], higher chance of post-surgery pneumonia [40] and infection [41] as well as multi-organ-failure [42]. It is well known that stored RBCs will gradually lose the allosteric effector 2,3-bisphosphoglycerate (2,3-BPG) and ATP with increased storage time. This results in a variety of harmful changes to the function and structural integrity of RBCs preserved in vitro including an increase in O2 affinity of the O2 storage and transport molecule hemoglobin (Hb) contained inside the RBC [43, 44], alteration in shape from a biconcave disk into a spheroechinocyte [45, 46], vesiculation from the surface of the RBC [47] and loss of membrane integrity [48-50] as well as loss of RBC deformability [51, 52]. In general, these functional and structural changes (i.e. the RBC storage lesion) reduce the post-transfusion survival of administered RBCs [53]. Here,

RBC survival is defined as the percentage of RBCs that are still in circulation 24 hours after transfusion [54]. Currently in the US, the acceptable shelf-life of a unit of RBCs stored at 4°C in a blood bank is no longer than 42 days [21]. In light of the RBC storage lesion, there is concern that RBCs stored for shorter periods of time (i.e. less than 42 days) are more efficacious than RBCs stored at the point of outdate (i.e. 42 days).

1.1.3 Limited supply of blood

The supply of human blood in the US is steadily shrinking while the demand of blood is expected to rise [43]. More than 50% of the recipients of blood transfusions are over the age of 65 in the US and this segment of the population is expected to double during the next 20 years [55, 56]. Thus, due to aging of the donor population and the increase in patients who need RBC transfusions [57], the US is expected to have a shortage of 4 million units of blood by the year 2030 [58]. Moreover, the global distribution and blood banking of some blood types is unbalanced. For example, the supply of some rare RBC types are rather limited in certain regions such as Rh negative

RBCs in Asia (<2%) [59]. To further compound the problem, the blood supply can be even more limited in emergency situations such as wars or natural disasters [60].

Considering these drawbacks associated with RBC transfusion and the possibility of future blood shortages, it has been a long-term goal of scientists and engineers to develop a universal readily-available O2 carrying solution for use in transfusion medicine. In this dissertation, we plan to synthesize a new generation of hemoglobin (Hb)-based O2 carrier

(HBOC) with fewer side-effects, longer circulation time and better oxygenation ability.

1.2 Types of artificial O2 carriers

Currently, depending on the type of O2 transport mechanism involved, artificial O2 carriers can be subdivided into two groups: 1) perfluorocarbon (PFC)-based O2 carriers

(PFCOCs) in which O2 is physically dissolved in the PFC phase and 2) Hb-based O2 carriers (HBOCs) in which O2 is covalently bound to the heme group of Hb.

1.2.1 PFCOCs

PFCs are linear, cyclic or polycyclic organic molecules in which all the hydrogen atoms are replaced by fluorine atoms [61]. Due to the low polarizability of fluorine atoms,

PFCs have very weak van der Waals interactions between neighboring molecules and behave like gas-like fluids [43, 61, 62]. Consequently, PFCs have the highest dissolving capacity for gases such as O2, CO2, NO, etc [43]. In addition, not only does the carbon- fluorine single bond in PFCs possess a very high bond energy of 530 KJ/mol, which is one of the strongest among molecular compounds, but also the carbon-carbon single bond energy of PFCs is enhanced because of the electron-attracting character of the fluorine atoms [62]. Thus, PFCs have exceptional chemical stability and inertness among organic molecules and also cannot be oxidized or metabolized by humans or microorganisms [62].

These properties make PFCs promising O2 carriers. Two commercial PFCOCs products:

Fluosol® (Fluosol-DA Green Cross Corp., Osaka, Japan and Alpha Therapeutic Corp.,

Los Angeles, CA) and Perftoran® (Perftoran Corp., Russia) have been approved for specific medical applications in US, Russia and Mexico, respectively [61, 63]. Currently, several commercial PFCOCs are under clinical investigation by the US Food and Drug

Administration (FDA) such as Oxygent® (Alliance Pharmaceutical Corp., San Diego,

CA), Oxyflour® (HemaGen, St. Louis, MO) and Oxycyte (Oxygen Biotherapeutics, Inc.,

Morrisville, NC) [61].

® Figure 1.1: Comparison of the O2 carrying capacity between whole human blood and the PFC Oxygent [1].

However, the weak van der Waals interactions between PFC molecules make PFCs extremely hydrophobic. Thus, PFCs must be emulsified with biocompatible amphiphiles such as lipids before intravascular administration. It has been challenging to find a PFC that is easy to excrete, emulsify and manufacture at low cost [62]. Moreover, in contrast to Hb encapsulated inside RBCs, the solubility of O2 in PFCs is linearly dependent on the pO2 (partial pressure of O2), see Figure 1.1 [1]. Therefore, not only is the O2 carrying capacity of PFCs much lower than that of Hb, but also much of the O2 carried by PFCs will be released before it reaches the capillaries where O2 is required by the surrounding tissues. In addition, administrated PFCs will activate macrophages and be cleared from the circulation by the reticuloendothelial system (RES), leading to the development of flu-like symptoms [64] e.g., fever and nausea, enlargement of the liver and spleen due to the accumulation of PFCs in tissues [65, 66], as well as transient inhibition of RES clearance function at high administration doses [43]. These problems may have hampered

6 the clinical use of PFCOCs [61, 67] and lead to the suspension of phase III clinical trials of Oxygent® and Oxyfluor® [68, 69]. Moreover, the production of Fluosol® was ceased due to its limited intravascular half-life, poor stability and complicated preparation [62,

63, 70].

1.2.2 Background on Hb

Figure 1.2: A: The structure of human Hb (PDB 1GZX) [2] and B: The structure of the heme group [3].

Because of its innate gaseous storage and transport functions, Hb is a natural precursor for artificial O2 carrier development. In the RBC, Hb is the most prevalent protein present [71] and is responsible for storage and transport of O2 and other gaseous ligands [72]. Under normal physiological conditions, Hb binds more than 98% of O2 in vivo [73] and can increase the human arterial blood oxygen concentration from 130 μM

7 to 8630 μM [74]. Administration of purified Hb is exempt from the immunological cross matching issues associated between donors and recipients [75]. Therefore, it is logical to explore stroma-free Hb as a potential RBC substitute. Structurally, Hb is a tetrameric protein comprised of two pairs of polypeptides chains: two α globin chains and two β globin chains (Figure 1.2.A) [72]. Each globin chain contains a heme prosthetic group, which is a cyclic tetrapyrrole with an iron atom at the center of the ring which can reversibly bind one molecule of O2 (Figure 1.2.B) [72]. Therefore, one Hb molecule can reversibly bind 4 molecules of O2.

Figure 1.3: A: The O2 equilibrium curve of Hb; B The transition of Hb between the T- state (PDB 4HHB) [4] and the R-state (PDB 1HHO)[5].

When no O2 is bound to the 4 heme groups, the quaternary conformation of deoxygenated Hb (deoxyHb) is maintained in the tense (T) state, which is characterized by its low O2 affinity and by the presence of salt bridges between the carboxyl terminals of all four globin subunits [72, 76]. Oxygenation of Hb (oxyHb, i.e. all 4 heme groups have O2 bound to them) successively leads to the rupture of these salt bridges and

8 increases the O2 affinity of Hb, transforming the quaternary conformation of Hb into the high O2 affinity relaxed (R) state [77]. Hb can shift between the low O2 affinity T-state

(i.e. deoxyHb) and the high O2 affinity relaxed R-state (i.e. oxyHb) when O2 binds or dissociates from Hb (Figure 1.3.B). Thus, O2 binds to Hb in a cooperative manner

(Figure 1.3.A) allowing Hb to effectively bind O2 in the lungs where the pO2 is high

(105-110 mm Hg) and release O2 to tissues and organs where the pO2 is low (<40 mm Hg)

[78].

The O2 affinity of Hb is described by the P50, which is the pO2 at which the Hb is half-saturated with O2. A high P50 value is indicative of low O2 affinity Hb while a low

P50 value is indicative of high O2 affinity Hb. For comparison, the P50 of freshly drawn human RBCs is ~ 26±1 mm Hg [79].

Inside the RBC, the iron atom in Hb is primarily kept in the 2+ valence state (ferrous state) by the combined effect of a series of intracellular reducing enzymes such as superoxide dismutase (SOD) [80, 81], catalase [82] and methemoglobin (metHb) reductase [83]. Outside the RBC, the Fe atom in Hb which is initially in the 2+ valence state can be easily oxidized into the 3+ valence state (ferric state) transforming Hb into metHb, which is incapable of binding or transporting O2 [72]. MetHb can induce the formation of reactive O2 species (ROS) [84], which can damage lipids, proteins and nucleic acids [85-87]. It is also structurally unstable and can unfold, thereby releasing the cytotoxic heme group [3, 43]. Therefore, the metHb level of an acceptable O2 carrier should be less than 10% in order to maximize its O2 carrying capacity and reduce its detrimental aspects [88].

9 1.2.3 HBOCs:

1.2.3.1 Stroma-free Hb as an HBOC

In light of the gaseous storage and transport ability of Hb contained within RBCs, purified Hb was directly administrated in vivo as an HBOC [89, 90] in early attempts to treat hemorrhage. However, intravenous transfusion of acellular Hb resulted in two major side-effects [84, 91-97].

First, Hb is toxic to tissues. Tetrameric Hb (22) easily dissociates into two pairs of

 dimers [91, 92] in the circulation especially when oxygenated , which are extremely prone to oxidation [97] and enhanced renal excretion [91, 98, 99]. The renal excretion of

Hb dimers can cause renal tubule obstruction, acute renal failure and promote the short circulation half-life of Hb in vivo [91, 98, 99]. The process of Hb oxidation to metHb promotes unfolding of the globin chains and releases cytotoxic heme into the systemic circulation also contributing to the aforementioned kidney tubule damage and renal failure [91, 92]. The hydrophobic heme molecule can diffuse into the phospholipid bilayer of cell membranes to induce lipid oxidative damage [43, 100] leading to cell lysis and death [101] as well as hemolysis of RBCs in vivo [3]. The Fe2+ contained in the free heme can be readily oxidized into Fe3+, turning heme into hemin [3]. Hemin can both damage mitochondrial DNA of hepatocytes to induce cell apoptosis [102] and directly induce degradation of genomic DNA [103]. In addition, in vitro studies have found that free heme can stimulate the expression of proinflammatory factors such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial leukocyte adhesion molecule (E-selectin), which may result in the local enrichment of leukocytes and induction of inflammation-related cell injury [104, 105]. 10 •- The oxidation of Hb to metHb can also generate the superoxide anion (O2 ) [106], which can react with other Hb molecules to form hydrogen peroxide (H2O2) and more metHb

• [43]. H2O2 can react with Hb in a Fenton-type reaction to produce hydroxyl radicals (OH ) and globin-centered ferryl radicals •HbFe4+=O. These ROS can initiate a series of oxidative cascades that can damage cell membranes, oxidize nucleic acids and proteins

[3].

Secondarily, the presence of extracellular Hb in the circulatory system can also elicit vasoconstriction and systemic hypertension. This is thought to occur via two mechanisms

[93-95, 107]. The first hypothesis suggests that free Hb tetramers and its dimers can extravasate through the endothelial cell layer of blood vessel walls and react rapidly with nitric oxide (NO) [108] via the NO dioxygenation reaction to form nitrate and metHb so as to inhibit NO-mediated activation of guanylate cyclase and vascular dilation [109]. It has also been observed that acellular Hb can activate platelets to release serotonin, which can induce the contractile response of injured arterial vessels by activating the Rho- associated kinase pathway in smooth muscle cells [110-112]. Besides, ferryl Hb, the oxidized form of Hb induced by H2O2, can convert inactive angiotensin I to active angiotensin II [113], which plays an important role in the hypertensive response [114].

The other hypothesis suggests an ―autoregulatory‖ response in which extracellular Hb facilitates O2 transport to the surface of the blood vessel wall and overoxygenates surrounding tissues, thereby eliciting vasoconstriction in order to reduce blood flow [95,

96]. An increase in the local pO2 coupled with scavenging of endothelium derived NO in small vessels can increase the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE)

[115], which plays a role in an autoregulatory mechanism that can increase the extent of

11 vasoconstriction. Consequently, both mechanisms of action also induce systemic hypertension. Regardless of the exact mechanism for the development of vasoconstriction and systemic hypertension, stroma-free Hb must be modified in order to eliminate or reduce the aforementioned adverse effects.

1.2.3.2 Strategies to modify Hb

Numerous strategies have been proposed to prevent Hb from dissociating into  dimers and reduce the harmful direct interactions between Hb with the blood vessel wall,

NO and surrounding tissues while preserving the O2 storage and transport capability of

Hb. These methods include intramolecular cross-linking of Hb, intermolecular cross- linking of Hb (i.e. Hb polymerization), conjugation of molecules to the surface of Hb, encapsulation of Hb inside particles and site-directed mutagenesis of Hb.

The intramolecular cross-linking strategy focuses on eliminating the renal damage resulting from dissociation of the Hb tetramer into two pairs of  dimers by covalently connecting neighboring globin subunits in the Hb tetramer to prevent its dissociation into dimers. Bis (3,5-dibromosalicyl) fumarate (DBBF) was previously employed to cross- link the α chains [116, 117] or β chains [118] of tetrameric Hb. Intramolecular cross- linking not only successfully prevented the rapid dissociation of the Hb tetramer into dimers, but also modified the O2 affinity of the cross-linked Hb [119, 120]. A commercial product HemAssist® (Baxter Healthcare, Deerfield, IL) was developed based on α-α- cross-linked Hb. However, although HemAssist® alleviated the renal toxicity elicited by acellular Hb, further development of this product was discontinued since serious adverse effects such as brain and pulmonary edema, pancreatic insufficiency, myocardial 12 ischemia [121] and high mortality [122] were observed in clinical trials. This was thought to be caused by rapid HBOC-induced NO scavenging at the injury site(s), which accelerated platelet deposition leading to increased vascular resistance [123-125].

Intermolecular cross-linking of Hb is aimed at increasing the size of the Hb molecule in order to prevent its rapid renal filtration and prolong the plasma retention time since molecules larger than 7 nm [126] are expected to extravasate less readily [43]. In previous studies, oxidized raffinose (O-raffinose) [127], 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide) (EDCI) [128, 129], difunctional reagents such as glutaraldehyde [130-132] and glycolaldehyde [133, 134] and tetrafunctional reagents such as N,N’-Bis(isophthalyl)fumarate [135] have been utilized to synthesize polymerized Hb (PolyHb) and ―bis-tetramer‖ style cross-linked Hb. In addition, Hb has been copolymerized with reductases such as SOD and catalase [136, 137]. The PolyHb- enzyme complex has beneficial antioxidant activity and low metHb levels. A rat ischemia-reperfusion model showed that the PolyHb-enzyme conjugate not only elicited less brain-blood barrier disruption and brain edema, but also was effective in reducing level of ROS after transfusion of HBOC [136, 138]. Thus, although no clinical investigation of these products has been reported in the literature [43, 68], this strategy appears promising. Several commercial products based on PolyHb such as Hemolink®

[139] (O-raffinose polymerized human Hb, Hemosol, Toronto, ON, Canada), Oxyvita®

[129] (―zero-linked‖ polymerized bovine Hb, OxyVita Inc, New Windsor, NY),

PolyHeme® [140] (pyridoxalated glutaraldehyde polymerized human hemoglobin,

Northfield Laboratories Inc., Evanston, IL) and Hemopure® [141] (glutaraldehyde polymerized bovine Hb, Biopure Corp., Cambridge, MA) have been developed for

13 clinical investigation. Hemopure® has been approved for clinical use in South Africa

[141]. However, extended clinical use of these products is still hampered by vasoactivity

[142-145] and cardiac toxicity [146]. Therefore, there must be further research to engineer the next generation of PolyHbs to reduce/prevent these side-effects.

Conjugation of biocompatible macromolecules such as dextrans and poly(ethylene glycol) (PEG) to the surface of Hb comprises another approach to decrease the side- effects associated with transfusion of Hb [147-151]. This strategy is useful in increasing the MW and size of Hb. A maleimide-activated PEG conjugated Hb, MP4, has been demonstrated to yield a long circulation time and no vasoconstriction in animal studies

[107, 150, 152, 153]. A draw back of this approach is the fact that conjugation of PEG is expensive to implement, which limits the scale-up potential of this strategy.

Encapsulation of native Hb inside the aqueous core of vesicles represents a simple strategy for engineering a structural mimetic of an RBC [154]. The most widely used vesicles consist of liposomes composed of biocompatible phospholipids, cholesterol, and

PEG-conjugated lipids [155, 156]. Liposome-encapsulated Hb (LEHb) particles maintain the O2 storage and transport functions of Hb prevent Hb from interacting directly with blood vessel wall and are expected to prolong the circulation time in vivo and reduce Hb dissociation (due to the high internal Hb concentration within the aqueous core of the vesicle), extravasation, renal toxicity and vasoconstriction [157-160]. However, LEHb particles are primarily cleared from the blood via the reticuloendothelial system (RES) and therefore accumulate in the liver and spleen [155, 156, 159], which saturates the RES with lipid and weakens the immune system. To address this concern, biodegradable diblock copolymers composed of PEG and either poly(caprolactone) (PCL) or

14 poly(lactide) (PLA) have been used to formulate polymersome-encapsulated Hb in which the copolymer membrane can degrade into harmless byproducts which can be easily cleared from the body [158-160]. Commercial LEHb products such as Hb-vesicles (HbV,

Waseda University and Keio University, Japan) and Neo Red Cells (Terumo Inc,

Kanagawa, Japan) were developed for clinical evaluation [157, 161-163]. However, the safety and efficacy of LEHbs is still under debate [164, 165]. In addition to lipid accumulation in the RES, a major drawback of Hb encapsulation is the high cost of lipids and amphiphilic copolymers used for vesicle formulation.

Site-directed mutagenesis of Hb represents another approach that is being used to prevent renal filtration of Hb and reduce NO scavenging of the Hb molecule while maintaining its oxygenation ability. Considering the ultra stability of the  interface

[166], the first site-directed mutagenesis strategy was focused on genetically engineering a α-α-fusion Hb tetramer which was physically unable to dissociate into  dimers [167].

Therefore, one of the first recombinant Hbs (rHbs) to be developed via this strategy consisted of rHb1.1 (Baxter Hemoglobin Therapeutics, Boulder, CO). Unfortunately, although rHb1.1 exhibited no renal toxicity, it elicited adverse side-effects such as high arterial blood pressure, decreased heart rate, fever and chills, which are related to NO scavenging [168, 169]. This preliminary work was followed up by the design of rHbs with low NO dioxygenation rate constants by multiple site-directed mutagenesis [170-

172]. Although rHbs with lower NO dioxygenation rate constants exhibited no hypertensive response, these proteins are prohibitively expensive to scale up.

15 1.2.4 Ultra high MW PolyHbs can address the side-effects of acellular HBOCs

Despite differences in the strategies that have been proposed to develop HBOCs, currently, it is well accepted that increasing the molecular radius of the HBOC via chemical modification can potentially mitigate these deleterious side-effects [95, 126,

150]. Therefore, polymerization of Hb with the difunctional cross-linking reagent glutaraldehyde [173] represents an easy-to-scale-up strategy that can yield large sized

PolyHbs that should be able to prevent the undesired extravasation/interaction of Hb through/with the blood vessel wall, thus prolonging the HBOC’s half-life [95, 174].

Glutaraldehyde has been widely employed to non-specifically cross-link/polymerize Hb

[175-177]. Although glutaraldehyde polymerization lacks site specificity [178], it does not induce structural modifications to critical functional regions of Hb. In contrast, polymerization with O-raffinose may alter βCys93 structurally [139], which plays an important role in the transport of NO [179], detoxification of superoxide ions [180] and allosteric regulation of Hb [181]. Thus, polymerization with glutaraldehyde does not induce harmful changes in Hb functionality and stability otherwise caused by O-raffinose polymerization [182, 183].

Two glutaraldehyde PolyHbs: Hemopure (HBOC-201, OPK Biotech, Cambridge,

MA) and PolyHeme have undergone phase III clinical trials. Hemopure® consists of polymerized bHb (PolyHb) with a P50 of 38 mm Hg and MW ranging from 130 to 500 kDa (average MW ~250 kDa) [21, 141, 184-186]. On the other hand, PolyHeme consists of a pyridoxylated polymerized human Hb (PolyhHb) with a P50 ranging from

28-30 mm Hg, and MW ranging from 128 to 400 kDa (average MW ~150 kDa) [140,

187, 188]. Despite commercial development of glutaraldehyde-polymerized Hbs, 16 hypertension and other important safety concerns remain critical impedances to clinical use of HBOC-201 and PolyHeme in the US [142-145, 185]. These safety issues indicate either the existence of Hb tetramer or oligomers in the PolyHb solution which are capable of extravasating through the blood vessel wall and scavenging NO, or that these HBOCs are not large enough to reduce the rate of facilitated O2 diffusion to the blood vessel wall.

Therefore, although glutaraldehyde polymerization is a feasible method to produce

HBOCs, there still exists a substantial need to synthesize larger sized PolyHbs to alleviate or eliminate the vasoconstriction and hypertension elicited by these commercial products.

In previous work conducted by our group, we quantitatively investigated the effect of glutaraldehyde stoichiometry on the degree of Hb polymerization and reported that the

MW of the resulting PolyHbs increased proportionally to the cross-link density (i.e. molar ratio of glutaraldehyde to Hb [G:Hb]) [130]. However in these early studies,

PolyHbs were not synthesized in an exclusive quaternary state [130]. In a recent study, we polymerized Hb exclusively in either a low or high oxygen affinity state at different cross-link densities [189]. Although we did not separate unpolymerized Hb from the

PolyHb mixture, we demonstrated control over the PolyHb MW and O2 affinity[189].

Therefore, we demonstrated that it is possible to synthesize high MW PolyHbs at high cross-link densities. In another study, we fractioned a L-PolyHb mixture into two fractions: one above 500 kDa in MW and one below 500 kDa in MW and observed that the fraction above 500 kDa in size polymerized at a G:Hb of 50:1 caused no vasoconstriction and the elicited the lowest increase in the mean arterial pressure (MAP) compared to other PolyHb fractions examined in that study [190]. In contrast, the vasoactivity of the PolyHb fractions >500 kDa in size at lower cross-link densities was

17 inversely proportional to the PolyHb MW. This result was consistent with Sakai’s observations that vasoconstriction and hypertension were inversely proportional to the size of the HBOC [174].

In addition, high MW PolyHb solutions will also possess high solution viscosities.

On the surface, this biophysical property of PolyHb solutions appears counterintuitive for a transfusion solution. However, there was the perception in the transfusion community that increasing the plasma viscosity from baseline levels was detrimental to blood circulation, and improved perfusion of O2 was expected to be achieved only by lowering the plasma viscosity [191]. In light of this assumption, the viscosity of first generation

PolyHbs such as Hemopure® (1.3 cP [184]) and PolyHeme® (1.9-2.2 cP [192]) was engineered to be much lower than that of blood (~3 cP [193]). However, low viscosity blood fails to transfer enough pressure to the capillaries, thus leading to a decrease in the functional capillary density (FCD) [191], which is the number of capillaries exhibiting transiting RBCs per unit area of tissue [194]. The FCD is independent of oxygenation of the tissues [195] but is a linear function of capillary pressure [196] and is considered to be the principal determinant of the survival of recipients of transfusion solutions after hemorrhagic shock [195, 197]. Thus, the development of a new generation of HBOCs with higher viscosities than that of blood may be beneficial for use in transfusion medicine. It has been found that hyperviscous volume expander solutions consisting of high MW colloidal molecules can maintain the FCD by exerting pressure on capillaries and increasing blood vessel wall shear stress [195, 197] so as to induce the endothelium to produce the vasodilator NO and prostaglandin [198-203]. This shear stress-dependent

18 release of vasodilators can offset the NO scavenging effect elicited by acellular HBOCs to some extent and increase the blood flow in the microcirculation.

In summary, high MW PolyHbs, which are synthesized in either the low or high O2 affinity state and absence of low MW Hb oligomers, tetramers and dimers, can potentially reduce the extent of vasoconstriction and hypertension upon transfusion. This approach is simple to implement, easy to scale up and takes advantage of the enormous amount of developmental data already available on PolyHb solutions.

1.3 Objective of Dissertation

The objective of this dissertation is to synthesize and characterize a small library of variable MW glutaraldehyde PolyHbs with two distinct O2 affinities. We hypothesize that

HBOC size will regulate vasoconstriction in the microcirculation, systemic hypertension as well as oxidative damage to tissues and organs. Increasing the size of the PolyHb can decrease both its ability to extravasate through the pores in the blood vessel wall and scavenge NO near the endothelium. It can also decrease the magnitude of the PolyHb diffusion coefficient, which will reduce the rate of PolyHb transport to the blood vessel wall and therefore dually reduce the rates of NO scavenging and HBOC-facilitated overtransport of O2. In addition, there is a lot of debate in the blood substitute research community about the effect of O2 affinity on O2 delivery in vivo, vasoactivity and hypertension [204-206]. Thus, the O2 affinity of PolyHb in this project will be engineered by synthesizing PolyHb with both low O2 affinity (L-PolyHb) and high O2 affinity (H-

PolyHb).

19 The starting material utilized in this project consists of purified bovine Hb (bHb) instead of stroma free human Hb as the starting material for PolyHb synthesis, since the

- O2 affinity of bHb is regulated by Cl ions instead of the allosteric effector 2,3-BPG [182].

In chapters 2 and 3, a series of variable MW PolyHb solutions was synthesized and characterized both in vitro and in vivo. More specifically, chapter 2 investigated the biophysical properties and pharmacokinetics of ultrahigh MW PolyHb solutions maintained in either the T- or R-state during the polymerization reaction. In chapter 3, the effect of varying the G:Hb molar ratio on the biophysical properties of PolyHb solutions polymerized in either the low or high O2 affinity state was investigated. In addition, the ability of these PolyHbs to potentially oxygenate tissues in vivo was preliminarily evaluated by an O2 transport model to simulate O2 transport in a hepatic hollow fiber bioreactor. In chapter 4, the effect of shear-stress induced NO production on NO transport in the microcirculation due to the high viscosity of PolyHb solutions was investigated by a combined NO and O2 transport simulation model in an arteriole. This model exhibited an improvement in NO transport by viscous PolyHb solutions upon transfusion. In chapter 5, we will outline future work on PolyHb solutions.

Taken together, the information gained from this study can be used to guide the design of the next generation of HBOCs for use in tissue engineering and transfusion medicine applications.

20 CHAPTER 2

Synthesis, Biophysical Properties and Pharmacokinetics of Ultrahigh MW PolyHbs

2.1 Introduction

As mentioned in the introductory chapter, polymerization of Hb with the difunctional cross-linking reagent glutaraldehyde, which can non-specifically react with numerous surface amino acid groups of Hb [207], represents a simple strategy to synthesize HBOCs.

Using this approach, it is possible to increase the molecular size of the HBOC by increasing the cross-link density. Therefore, preventing the harmful dissociation of Hb tetramers into αβ dimers and prolonging the circulation time of these molecules in the blood compartment [131, 146, 176]. In light of this approach, Hemopure is the only glutaraldehyde PolyHb approved for clinical use in South Africa [141].

Recent late stage clinical results of PolyHb solutions hamper clinical utilization in the US [142-144]. The resultant safety issues may be attributed to vasoactivity and/or oxidative events caused by the PolyHb solutions. The interactions of Hb with the vascular endothelium or sub-endothelium can occur with certain modified Hbs either by extravasation through or interaction with the vascular endothelium. Interactions may include NO scavenging, increased facilitated diffusion of O2 to surrounding tissues and oxidative side reactions at the surface of the endothelial layer or within sub-endothelial

21 compartments. Therefore, increasing the molecular size of the HBOC is expected to be able to mitigate the aforementioned side-effects of acellular HBOCs [95, 150]. An acceptable HBOC should have a diameter of at least 7 nm in order to prevent extravasation through blood vessel walls [126] and reduce the facilitated diffusion of O2 to surrounding tissues [95]. In addition, tetrameric Hb should not be present in the

PolyHb solution in order to reduce/eliminate vasoactivity [208]. With this in mind, the next generation of PolyHbs should be synthesized with larger MWs and contain no free tetrameric Hb in solution compared to HBOC-201® and PolyHeme.

To our knowledge, high MW glutaraldehyde PolyHbs constrained in an exclusively low or high O2 affinity state have never been synthesized. In comparison, the MW of both Hemopure® and PolyHeme® are less than 500 kDa [140, 141]. Recently, we demonstrated control over the PolyHb’s O2 affinity (P50) and absolute MW distribution by polymerizing Hb exclusively in either the low (L-PolyHb) or high (H-PolyHb) O2 affinity state at different cross-link densities [209]. In another study, we separated L-

PolyHb mixtures that had been polymerized at different cross-link densities into two fractions: one above 500 kDa in MW and another below 500 kDa in MW [190] and observed that the PolyHb fraction above 500 kDa in MW with the highest cross-link density (50:1) yielded no vasoconstriction and the lowest increase in the mean arterial pressure compared to other PolyHb fractions examined in this study [190]. The results from this previous study support Sakai et al.’s observations, in which it was shown that vasoconstriction and hypertension were inversely proportional to the size of the HBOC

[174].

22 Therefore, synthesis/formulation of HBOCs with large molecular sizes may enhance

Hb compartmentalization within the vascular space, extend exposure times and limit vasoconstriction/hypertension. This new design approach satisfies the two potential mechanisms mentioned in chapter 1 for the development of vasoconstriction and hypertension upon administration of HBOCs and may optimize circulation times for extended duration therapeutic applications.

In this chapter, purified bovine Hb (bHb) was polymerized with glutaraldehyde at high glutaraldehyde:Hb (G:Hb) molar ratios in both the low and high O2 affinity states to prepare ultrahigh MW PolyHbs with distinctly different O2 affinities. The PolyHbs were then fractioned via tangential flow filtration (TFF) to obtain the fraction above 500 kDa in MW and underwent subsequent buffer exchange with a modified Ringer’s buffer. The ultrahigh MW PolyHb solutions were then evaluated for various biophysical, rheological, pharmacokinetic properties such as their MW distribution, O2 affinity, cooperativity coefficient, viscosity, colloid osmotic pressure (COP), gaseous ligand binding/release kinetics, as well as their in vitro/in vivo oxidative tendencies.

2.2 Materials and Methods

2.2.1 Materials

Glutaraldehyde (70%), NaCl, KCl, NaOH, Na2S2O4, NaCl (USP), KCl (USP),

CaCl2-2H2O (USP), NaOH (NF), sodium lactate (USP), N-acetyl-L-cysteine (USP),

NaCNBH3 and NaBH4 were purchased from Sigma-Aldrich (Atlanta, GA). Sephadex G-

25 resin was purchased from GE Healthcare (Piscataway, NJ). KCN, K3Fe(CN)6, and all other chemicals were purchased from Fisher Scientific (Pittsburgh, PA).

23 In preparation for experiments, all glassware and plasticware were immersed in 1 mol/L NaOH solution for more than 6 hours to degrade any endotoxin present, followed by thorough rinsing with HPLC grade water.

2.2.2 Hb Purification

Fresh bovine blood stored in a 3.8% sodium citrate solution at a final concentration of 90:10 v/v (bovine blood:sodium citrate solution) was purchased from Quad Five

(Ryegate, MO). bHb was purified from lysed bovine RBCs (bRBCs) via TFF [210, 211]. bRBCs were initially washed 3 times with 3 volumes of isotonic saline solution (0.9%) at

4C. bRBCs were subsequently lysed on ice with 2 volumes of hypotonic, 3.75 mM phosphate buffer (PB) at pH 7.4 for 1 hour. The RBC lysate was then filtered through a glass column packed with glass wool to remove the majority of cell debris. Clarified bRBC lysate was then sequentially passed through 50 nm and 500 kDa hollow fiber cartridges (Spectrum Labs, Rancho Dominguez, CA) in order to remove additional cell debris and impurity proteins. The purified bHb was collected and concentrated on a 100 kDa hollow fiber cartridge (Spectrum Labs) to yield the raw material for synthesis of

PolyHb solutions.

2.2.3 Polymerization of Hb

L-PolyHb was synthesized according to a previously described procedure in the literature [189, 190]. To generate fully deoxygenated or tense (T)-state bHb, 30 grams of purified bHb was diluted with ice cold PB (20 mM, pH 8.0) to yield 1200 mL of bHb solution. The bHb solution was placed inside an airtight bottle and connected to a

24 vacuum manifold. The entire system was kept below 4oC in an ice water bath. The bHb solution was then subjected to several cycles of vacuum and argon (Ar) purging to remove the majority of O2 from solution. After 4 hours of vacuum and Ar cycling, approximately 300 mL of ice cold Na2S2O4 solution (1.5 mg/mL) was titrated into the bHb solution with a syringe pump (Razel Scientific, St. Albans, VT), while the pO2 of the solution was simultaneously monitored using a RapidLab 248 (Siemens, Malvern, PA) blood gas analyzer until the pO2 of the bHb solution attained a value of 0 mm Hg. At this point, an additional 30 mL of 1.5 mg/mL Na2S2O4 solution was added to the T-state bHb solution to maintain the pO2 at 0 mm Hg during and after the polymerization reaction. A

30 mL syringe was used to titrate glutaraldehyde preequilibrated with Ar into the sealed glass bottle under continuous stirring. A 50:1 molar ratio of glutaraldehyde to bHb was used for the T-state bHb polymerization reaction.

Relaxed (R)-state bHb was prepared in a similar manner to T-state bHb using the same vacuum manifold system. 1500 mL of 20 mg/mL bHb solution was saturated with pure O2 for 2 hours in an ice-water bath and the pO2 was monitored using a RapidLab

248 blood gas analyzer. When the pO2 measured was well over the 749 mm Hg measurement range of the RapidLab 248 blood gas analyzer, a 30 mL syringe was used to titrate glutaraldehyde in the sealed glass bottle under continuous stirring. A 40:1 molar ratio of glutaraldehyde to bHb was used for the R-state bHb polymerization reaction.

The resulting T- or R-state bHb solutions were then allowed to react with glutaraldehyde in the dark at 37oC for 2 hours, and were stirred and equilibrated with either pure Ar (50:1 T-state bHb) or O2 (40:1 R-state bHb). At the end of the 2 hour reaction period, 5 mL of 8 M NaCNBH3 in PB buffer (20 mM, pH 8.0) was injected into

25 the glass bottle to reduce the Schiff base and reduce the metHb level of the PolyHb solution. The PolyHb solution was continuously stirred for 30 min in an ice-water bath.

Subsequently, 20 mL of 2 M NaBH4 in PB buffer (20 mM, pH 8.0) was injected into the glass bottle to quench the polymerization reaction. The pO2 of the bHb solution before polymerization, after polymerization, and after quenching with NaBH4 was measured using a RapidLab 248 blood gas analyzer. All reactions were repeated in triplicate.

2.2.4 Clarification and Separation of PolyHb Solutions

Initially, each PolyHb solution was clarified by filtering it through a glass column packed with glass wool that had been autoclaved at 250oC for 30 min [212] in order to degrade any endotoxin present in the glass wool. The clarified PolyHb solution was then separated into two distinct MW fractions with a 500 kDa hollow fiber cartridge

(Spectrum Labs). The retentate mostly contained PolyHb molecules that were larger than

500 kDa. This fraction was used for all subsequent studies described in this work.

2.2.5 Desalting and Buffer Exchange of PolyHb solutions

After clarification and separation, the PolyHb was suspended in PB buffer along with reduced glutaraldehyde and excess NaBH4 and NaCNBH3. The PolyHb solution underwent buffer exchange to remove cytotoxic glutaraldehyde, NaCNBH3 and NaBH4

[213] with a modified lactated Ringer’s solution (NaCl (USP) 115 mmol/L, KCl (USP) 4 mmol/L, CaCl2-2H2O (USP) 1.4 mmol/L, NaOH (NF) 13 mmol/L, sodium lactate (USP)

27 mmol/L and N-acetyl-L-cysteine (USP) 2 g/L). The buffer exchange was conducted using an ÄKTA Explorer 100 system controlled by Unicorn 5.1 software (GE

26 Healthcare). An XK 50/30 (300 mm in length, 50 mm I.D.) column (GE Healthcare) was packed with 500 mL of Sephadex G-25 medium resin at room temperature. After equilibrating the column with modified lactated Ringer’s solution at a flow rate of 8 mL/min, the PolyHb solution was injected into the XK 50/30 column via a superloop (50 mL, GE Healthcare) at a flow rate of 5 mL/min. 100 mL of sample was loaded onto the column each time and then eluted with the modified lactated Ringer’s solution. The protein concentration was detected at a wavelength of 280 nm, while the salt concentration was monitored with a conductivity detector. During the buffer exchange process, the UV signal increased as PolyHb eluted from the column, while the conductivity decreased when reduced glutaraldehyde and NaBH4 (and NaCNBH3 for H-

PolyHb) eluted from the column. The buffer exchanged PolyHb solution was collected as the UV signal increased, but before the conductivity signal decreased. The PolyHb fraction was then concentrated with a 100 kDa hollow fiber cartridge (Spectrum Labs) and stored at -80°C.

2.2.6 MetHb Level and Protein Concentration of PolyHb Solutions

The metHb level of bHb/PolyHb solutions was measured via the cyanomethemoglobin method [214]. Total protein concentration was measured using the

Bradford method [215] using the Coomassie Plus protein assay kit (Pierce Biotechnology,

Rockford, IL).

27 2.2.7 SDS-PAGE of PolyHb Solutions.

The MW distribution of bHb/PolyHb solutions was initially assessed via gel electrophoresis using a Mini-PROTEAN 3 Cell (Bio-Rad; Hercules, CA). All samples were mixed with an equal volume of sample buffer (Bio-Rad) containing 5% v/v β- mercaptoethanol, and then boiled for 5 min. A 4% stacking gel with a 12% resolving gel was assembled on a minivertical gel apparatus and each lane was loaded with 25 µg of protein. The gel was run at 120 V for approximately 1 hour. After electrophoresis, the gel was stained with Coomassie blue R250 (stain buffer, Bio-Rad) for one hour and then destained with a buffer consisting of 10% acetic acid and 20% methanol. The gel was scanned on a Gel Doc XR (Bio-Rad) imaging system for further analysis.

2.2.8 Size Exclusion Chromatography (SEC) Coupled with Multi-Angle Static Light

Scattering (MASLS) Analysis of PolyHb Solutions.

The absolute MW distribution of bHb/PolyHb solutions was measured using a SEC column (Ultrahydrogel linear column, 10 μm, 7.8×300 mm, Waters, Milford, MA) driven by a 1200 HPLC pump (Agilent, Santa Clara, CA), controlled by Eclipse 2 software

(Wyatt Technology, Santa Barbara, CA) connected in series to a DAWN Heleos (Wyatt

Technology) light scattering photometer and an OptiLab Rex (Wyatt Technology) differential refractive index detector. The mobile phase consisted of 20 mM PB (pH 8.0),

100 ppm NaN3, and 0.2 M NaCl (Fisher Scientific) in HPLC grade water that was filtered through a 0.2 μm membrane filter. PolyHb solutions were diluted with the mobile phase, and 60 μL of sample was injected onto the column via a 1200 Autosampler (Agilent). All data were collected and analyzed using Astra 5.3 (Wyatt Technology) software.

2.2.9 O2-PolyHb Equilibria.

The O2 affinity and cooperativity coefficient of bHb/PolyHb solutions were regressed

from O2-PolyHb equilibrium curves measured on a Hemox Analyzer (TCS Instruments,

Southampton, PA) at 37oC.

Samples were prepared by thoroughly mixing 100 μL of sample with 5 mL of

Hemox buffer (pH 7.4, TCS Instruments), 20 μL of Additive-A, 10 μL of Additive-B and

10 μL of anti-foaming agent to the heme concentration of about 60 μM. The bHb/PolyHb

sample was allowed to equilibrate to a pO2 of 145±2 mm Hg using compressed air. After

equilibrating the sample for 45 minutes, the gas stream was switched to pure N2 to

deoxygenate the bHb/PolyHb sample. The absorbance of oxy- and deoxy-Hb in solution

was recorded as a function of pO2 via dual wavelength spectroscopy. O2-PolyHb

equilibrium curves were fit to a four-parameter (A0, A∞, P50, n) Hill model [216]

(Equation 1). In this model, A0 and A∞ represent the absorbance at 0 mm Hg and full

saturation, respectively. The cooperativity coefficient is represented by the parameter n,

and the pO2 at which the bHb/PolyHb is half-saturated with O2 is represented by the O2

affinity or P50.

n Abs A0 pO2 Y   nn (1) AA  0 pO2 P 50

2.2.10 Stopped Flow Kinetic Analysis of PolyHb Solutions.

The rapid kinetics of gaseous ligand reactions with bHb/PolyHb was measured in an

Applied Photophysics SF-17 micro-volume stopped-flow apparatus as previously 29 described in the literature [217]. bHb/PolyHb solutions (30 M) were rapidly mixed with equal volumes of 1.5 mg/mL sodium dithionite (British Drug House, Poole, England), and O2 dissociation was monitored by absobance changes at 437.5 nm in 0.05 M bis-Tris buffer, pH 7.4. Multiple kinetic traces were averaged for each reaction, and fit to exponential equations using Marquardt-Levenberg fitting routines in the Applied

Photophysics software. The kinetics of CO binding with deoxygenated Hb/PolyHb solutions were measured at 437.5 nm in 50 mM bis-Tris buffer at pH 7.4 at 25C in the presence of sodium dithionite. The CO stock solution (about 1 mM) was prepared by saturating the degassed buffer solution with the flow of pre-washed CO gas.

The kinetics of NO oxidation with oxy-bHb/PolyHb solutions was carried out in the stopped-flow instrument as previously described in the literature [131]. NO stock solutions (∼2 mM) were prepared by saturating deoxygenated 0.05 M Tris buffer, pH 7.4, in a gas-tight serum bottle with NO gas that was pre-washed with deoxygenated 1 M

NaOH and buffer solutions. The NO stock solution was then transferred with a Hamilton syringe into a gastight syringe containing deoxygenated buffer solution to make appropriate concentrations of NO solutions. Hb/PolyHb solutions (1 M) were mixed with NO solutions (≤50 μM), and the absorbance changes of the reaction were followed at 420 nm. Multiple traces were averaged for each reaction, and fit to exponential equations to obtain reaction rate constants.

30 2.2.11 PolyHb Viscosity and COP.

PolyHb viscosity was measured in a cone and plate viscometer DV-II plus with a cone spindle CPE-40 (Brookfield Engineering Laboratories, Middleboro, MA) at a shear rate of 160/sec. The COP of PolyHb was measured using a Wescor 4420 Colloid

Osmometer (Wescor, Logan, UT) [218].

2.2.12 RBC Aggregation in PolyHb Solutions.

The extent of RBC aggregation in PolyHb solutions under stasis was measured using a transparent cone-plate shearing instrument that uses the light transmission method [219].

This instrument consists of a transparent horizontal plate and rotating cone between which the blood sample is placed, with a light source and photocell arranged vertically

(i.e., perpendicular to the plane of the cone and plate) to measure light transmission through the sample. The degree of RBC aggregation was assessed from triplicate measurements on a 0.35 mL sample of heparinized Syrian hamster blood mixed with the test solution at a volume ratio of 1:1, with the photometric rheoscope (Myrenne

Aggregometer, Myrenne, Roetgen, Germany). The Myrenne ―M‖ aggregation parameter is determined as follows: The sample is first exposed to a brief period of high shear (600 s−1) to disrupt any preexisting RBC aggregates. The rotation is then stopped, and the light transmittance through the blood sample is recorded for 10 s; the average change in light transmission over this period is taken as the M value (units are arbitrary). If no aggregation occurs, then the light transmission remains constant, and M = 0.

Aggregation of the RBCs reduces scattering and allows more light to reach the photocell,

31 yielding a positive M value, the magnitude of which increases with the degree of aggregation. The use of this technique as well as comparisons of this index of aggregation (M) with other methods and with different animal species has been described previously in the literature [220, 221]. M indexes in 5% human serum albumin (no aggregation) and 6% dextran 500 kDa (aggregation) were used as control solutions to compare with PolyHb solutions.

2.2.13 In Vitro Autoxidation of PolyHb Solutions.

All PolyHb solutions were converted to the ferrous (Fe2+) or oxy-PolyHb form immediately prior to autoxidation experiments. Experiments were carried out with 20-25

M heme in sealed cuvettes with room air equilibrated with 50 mM Chelex-treated potassium phosphate buffer at 37C. Absorbance changes in the range of 450-700 nm due to the spontaneous oxidation of Hb were recorded in a temperature-controlled photodiode array spectrophotometer (Hewlet Packard 8453, Palo Alto, CA). Similar oxidation assays were also performed in the presence of SOD (4.6 U/mL) and catalase (414 U/mL).

Autoxidation reactions were followed to near completion (~24 h), at which time 22 M potassium ferricyanide (K3Fe(CN)6) was added to completely oxidize the remaining ferrous PolyHb. A multicomponent analysis was performed to calculate the ferrous and ferric species based on their individual extinction coefficients. Autoxidation rates were obtained from plots of the loss of ferrous to ferric PolyHb versus time using nonlinear least-squares curve fitting (single-exponential, two parameter decay) techniques in

Sigma-Plot (SPSS, Chicago IL).

32 2.2.14 Animals and Surgical Preparation.

Male Hartley guinea pigs were purchased from Charles Rivers Laboratories

(Wilmington, MA) and acclimated for 1 week upon arrival to the FDA/Center for

Biologics Evaluation and Research (CBER) animal care facility. All animals were fed normal diets throughout the acclimation period and weighed 350-450 g at the time of study. Animal protocols were approved by the FDA/CBER Institutional Animal Care and

Use Committee with all experimental procedures performed in adherence to the National

Institutes of Health guidelines on the use of experimental animals.

On days of surgery, guinea pigs were anesthetized via the i.p. route with a cocktail of ketamine HCl (100 mg/kg) and xylazine HCl (5 mg/kg) (Phoenix Scientific In., St.

Joseph, MO). Under aseptic conditions a midline incision was made around the neck region allowing for blunt dissection and exposure of the right common carotid artery and the left external jugular vein. Saline filled catheters containing 50 IU of heparin per mL prepared from sterile PE50 tubing (Clay Adams of Becton-Dickenson, Sparks, MD) were placed in each vessel and tunneled under the skin to the back of the neck. Immediately following surgeries animals were administered a subcutaneous dose of ketoprofen (10 mg/kg) (Fort Dodge Pharmaceuticals, Fort Dodge, IA, USA) and allowed 24 hours of recovery prior to experimentation. Animals were randomized to receive a 20% blood volume exchange transfusion (ET) with either 50:1 L-PolyHb or 40:1 H-PolyHb (n=4 guinea pigs / group). Fully conscious and freely moving guinea pigs underwent a 20% ET replacing blood with PolyHb. Arterial and venous catheters were extended, tethered and connected to separate syringe pumps (Model 11 Harvard Apparatus, Holliston, MA) set on withdrawal (1 mL/min) and infuse (1 mL/min), respectively. The 20% ET volume in

33 the guinea pig was estimated using the equation described by Ancill R. J. [222] (20% ET

(mL) = (0.07 (mL/g) x body weight (g)) / 2). Plasma from blood in the heparinized withdrawal syringe for each transfused animal was obtained to determine the total

PolyHb removed during the exchange transfusion period (approximately 12 minutes).

Each PolyHb solution was transfused as a 100 mg/mL protein solution. Blood samples

(0.2 mL) were obtained from the arterial catheter prior to infusion (baseline) and at the end of ET (time 0), and at 0.25, 0.5, 1, 2, 4, 8, 12, 24 and 48 hours. Plasma was used for evaluation of: (1) total PolyHb; (2) ferrous PolyHb and (3) ferric PolyHb using a photodiode array spectrophotometer (Model 8453 Hewlet Packard, Palo Alto, CA) [223].

The circulating L- and H-PolyHb polymer distribution was evaluated as a function of time using a previously described method in the literature [224]. In brief, plasma samples

(50 L) were evaluated by size exclusion chromatography (SEC). Samples were run on a

BioSep-SEC-S3000 (600 mm x 7.5 mm) SEC column (Phenomenex, Torrance, CA) attached to a Waters Delta 600 pump and Waters 2499 dual-wavelength detector, controlled by a Waters 600 controller using Empower2 software (Waters Corp.,

Milford, MA).

2.2.15 Pharmacokinetic analysis of PolyHb Solutions.

The dose (mg) of PolyHb received by each animal at the end of ET was determined by subtracting the total amount of PolyHb in the plasma from whole blood collected in the ET syringe from the total amount of infused PolyHb according to the following equation (2.1):

dosereceived  ([PolyHb]inf used Vinf used )  ([PolyHb]totalET VET ) (2.1) 34 Where dose [PolyHb]infused is the concentration of PolyHb (mg/mL) infused, Vinfused is the PolyHb infusion volume (mL), [PolyHb ]totalET is the concentration of PolyHb

(mg/mL) from plasma sampled out of the withdrawal syringe and VET is the volume (mL) collected in the withdrawal syringe. Pharmacokinetic (PK) parameters were determined for total PolyHb, ferrous PolyHb (oxy/deoxy) and ferric PolyHb. Noncompartmental methods employed by WinNonlin version 4.1 (Pharsight Corp., Mountain View, CA,

USA) were used to calculate PK parameter estimates. The area under the plasma concentration time curve (AUC0-) was estimated using the linear trapezoidal rule to the last measurable concentration (AUC0-C last). Extrapolation to infinity (AUCC last-) was accomplished by dividing Clast by the negative value of the terminal slope (k) of the log- linear plasma concentration-time curve. Thus AUC0- is equal to the sum of AUC0-C last and AUCC last-. Additional parameters were calculated as follows: the plasma clearance

(CL) was calculated as the dose divided by AUC0-, the mean residence time (MRT) was

-1 calculated as k , the apparent volume of distribution (Vdss) was calculated as the product of CL and MRT and half-life (t1/2) was calculated as ln(2) divided by k.

2.3. Results

bHb was polymerized using glutaraldehyde as the cross-linking reagent at a G:Hb molar ratio of 50:1 for L-PolyHb and 40:1 for H-PolyHb. After polymerization, each

PolyHb mixture was fractionated with a 500 kDa hollow fiber cartridge (Spectrum Labs) to remove tetrameric Hb and Hb oligomers with MW less than 500 kDa. PolyHb fractions with MW above 500 kDa were used in this study.

35 2.3.1 pO2 of PolyHb Solutions.

Figure 2.1: pO2 at various stages of the bHb polymerization process for L- and H-PolyHb solutions. The error bar represents the standard deviation from triplicate reactions.

To ensure that Hb was polymerized in the T- or R-state, the pO2 at various stages of the bHb polymerization process was measured and shown in Figure 2.1. For L-PolyHb, the pO2 of the bHb solution was reduced to 0 mm Hg by argon purging and subsequent titration of Na2S2O4 before polymerization and remained at 0 mm Hg after the polymerization reaction and subsequent quenching with reducing agents. These results show that under the protection of an inert atmosphere, bHb was polymerized in an O2- free environment and maintained in the deoxygenated state (T-state) during the polymerization process. For H-PolyHb polymerization, the pO2 before and after polymerization were kept above the measurement range of the O2 detector. This showed 36 that bHb was saturated with O2 and bHb was maintained in the R-state during the polymerization reaction. After quenching the R-state bHb polymerization reaction with

NaCNBH3 and NaBH4, the pO2 of the H-PolyHb solution dropped to 0 mm Hg due to the reduction of O2 by NaBH4.

2.3.2 SDS-PAGE and MW Distribution of PolyHb Solutions.

Figure 2.2: SDS-PAGE of unmodified bHb, L- and H-PolyHb solutions.

Figure 2.2 shows the SDS-PAGE of unmodified bHb and high MW PolyHbs. Both

L- and H-PolyHb showed a strong band above 250 kDa and very weak bands around 15 and 30 kDa suggesting that L- and H-PolyHb are mostly polymerized by inter- and intramolecular cross-links. Therefore, these results showed the presence of very little  and 

37 monomers (these correspond to the two bands around 15 kDa, the lower band represents

 subunits while the upper band represents  subunits) as well as 2/2/ dimers in solution (these correspond to the band at 30 kDa), confirming that the TFF process was very effective in removing small MW Hb species less than 500 kDa in MW. The majority of both L- and H-PolyHb solutions were above 250 kDa in MW with the H-PolyHb being slightly larger (Figure 2.2).

Figure 2.3: Absolute MW distribution of unmodified bHb, L- and H-PolyHb solutions.

Light scattering results confirmed that H-PolyHb has a larger MW distribution compared to L-PolyHb (Figure 2.3). Despite this difference, both types of PolyHb solutions possess large MWs ranging from 16.59 to 26.33 MDa (260~400 bHb tetramers).

The light scattering results also indicate that there is no free Hb in the PolyHb solution,

38 since there is no peak corresponding to bHb tetramers. This shows that all bHb tetramers were polymerized in the reaction. However, SDS-PAGE results indicate that an extremely small fraction of the PolyHb solutions possess uncross-linked  and  monomers as well as 2/2/ dimers. While all the tetrameric Hb is polymerized, there are some  and  monomers as well as /2/2 dimers that are not cross-linked within the PolyHb superstructure.

2.3.3 P50 and n of PolyHb Solutions.

Figure 2.4: Equilibrium O2-bHb/PolyHb binding curves of unmodified bHb, L- and H-PolyHb solutions.

Figure 2.4 shows the equilibrium O2 dissociation curves of unmodified bHb, L- and

H-PolyHb. Compared to the O2-bHb equilibrium curve, the O2-PolyHb equilibrium curve

39 of L-PolyHb is shifted to the right, while the O2-PolyHb equilibrium curve of H-PolyHb is shifted to the left.

Figure 2.5: Oxygen affinity (P50) and cooperativity coefficient (n) of unmodified bHb, L- and H-PolyHb solutions. The error bar represents the standard deviation from triplicate reactions. ✴ p<0.05 with respect to unmodified bHb.

The regressed P50 and cooperativity coefficient (n) of bHb and fractionated PolyHb solutions are shown in Figure 2.5. The P50 of H-PolyHb is approximately 0.66 mm Hg, which is much lower than that of L-PolyHb which is approximately 41 mm Hg. The cooperativity coefficients of both L- and H-PolyHb solutions are less than 1.

40 2.3.4 MetHb Level of PolyHb Solutions.

Figure 2.6: MetHb level of unmodified bHb, L- and H-PolyHb solutions. The error bar represents the standard deviation from triplicate reactions. ✴ p<0.05 with respect to unmodified bHb.

The metHb level of unmodified bHb, L- and H-PolyHb is shown in Figure 2.6. The metHb level of unmodified bHb is very low (<1%), since it was purified from fresh bRBCs and stored at -80°C . In contrast, L- and H-PolyHb had similar metHb levels which were both below 4%.

2.3.5 Stopped Flow Kinetic Analysis of PolyHb Solutions.

Representative time courses for CO binding to deoxygenated bHb/PolyHb was obtained on the stopped-flow instrument and plotted in Figure 2.7 for comparison. While

CO binding to deoxygenated L-PolyHb is slightly slower compared to unmodified bHb, the binding of CO to deoxygenated H-PolyHb occurs much more rapidly. This is

41 supported by the CO concentration dependence of the apparent CO association reaction rates shown in the insert of Figure 2.7. Table 2.1 summarizes the kinetic parameters for

O2 dissociation from oxy-bHb/PolyHb, and the second order rate constants for CO association and NO dioxygenation with deoxygenated and oxygenated bHb/PolyHb, respectively. Both L- and H-PolyHb O2 dissociation rate constants deviated from that of unmodified bHb by either a 50% increase or 40% reduction, respectively. The main difference in gaseous ligand binding properties was observed in CO association to deoxygenated H-PolyHb, which resulted in a ~ 20-fold increase of the binding rate constant with respect to bHb. CO binding with L-PolyHb is only slightly lower than that of unmodified bHb. Conversely, the NO reaction remains largely unchanged among unmodified bHb, L- and H-PolyHb.

Figure 2.7: Rapid kinetics of CO binding with unmodified deoxygenated bHb, 50:1 L- and 40:1 H-PolyHb. Stopped-flow time courses of 15 M deoxygenated bHb/PolyHb (after mixing) reacting with 250 M CO solutions for unmodified bHb () and L-PolyHb (□) solutions, or 50 M CO solution for H-PolyHb (∆) were monitored at 437.5 nm in 50 mM phosphate buffer at pH 7.4 and room temperature. The solid lines are from the nonlinear least-square curve fitting of the time courses to the exponential equation. The obtained apparent rate constants were plotted versus CO concentration in the insert for each bHb/PolyHb to generate the second order rate constants of the reaction between deoxygenated bHb/PolyHb and CO (Table 2.1).

42 Table 2.1: Rapid kinetic parameters of gaseous ligand reactions with unmodified bHb, L- and H-PolyHb: O2 dissociation rate constant (koff, O2), CO binding rate constant (kon, CO), and the rate of NO dioxygenation (kox,NO).

-1 -1 -1 -1 -1 Sample koff, O2 (s ) kon, CO (µM s ) kox, NO (μM s ) Unmodified bHb 36.1 0.22 18.3 50:1 L-PolyHb 53.0 0.18 18.9 40:1 H-PolyHb 22.0 4.84 17.5

2.3.6 Viscosity and COP of PolyHb Solutions.

Table 2.2: The viscosity and COP of L- and H-PolyHb.

Concentration Viscosity COP Sample (g/dL) (cP) (mm Hg) 50:1 L-PolyHb 10 11.4 1 40:1 H-PolyHb 10 7.8 7 50:1 L-PolyHb 5 5.2 1 40:1 H-PolyHb 5 3.6 4

The viscosity and COP of PolyHb solutions is shown in Table 2.2. The viscosity of both PolyHb solutions increased with increasing PolyHb concentration. In contrast, the

COP of PolyHb solutions was fairly insensitive to PolyHb concentration. However, the

L-PolyHb solutions displayed lower COP versus H-PolyHb solutions.

2.3.7 RBC Aggregation of PolyHb Solutions.

The M index of aggregation of heparinized hamster blood mixed with PolyHb solutions is shown in Figure 2.8. The aggregation of RBCs induced by PolyHb solutions was significantly less than the aggregation induced by 6% dextran 500 kDa (a high viscosity plasma expander, with viscosity of 6.3 cP and COP of 38 mm Hg), but higher than the aggregation induced by 5% human serum albumin solution. The blood smears 43 show that mixtures of blood and 10% PolyHb generate minor rouleaux or rouleaux- rouleaux complexes compared to 6% dextran 500 kDa. The observed aggregation was very mild compared to dextran. The general trends for both PolyHb samples were very similar, both PolyHbs promoted static RBC aggregation compared to 5% albumin, however, significantly less than 6% dextran 500 kDa, both of them clinically used plasma expanders.

Figure 2.8: A typical blood smear for aggregation studies obtained from heparinzed hamster blood mixed in a 1:1 volume ratio with test solution. 5% albumin does not produce aggregation (above), however 6% dextran 500 kDa increases aggregation (below).

44 2.3.8 Pharmacokinetic Analysis of PolyHb Solutions.

1000

1.2

M) M) M)

  

( ( ( 1.0 800 Total heme 0.8 Fe2+ heme

Fe3+ heme

heme heme heme 0.6 600

0.4

Absorbance (AU) (AU) (AU) Absorbance Absorbance Absorbance Absorbance (AU) Absorbance 0.2 400 0 450 500 550 600 650 700 Wavelength (nm)

200

ConcentrationConcentrationConcentration

0 0 10 20 30 40 50 Time (hours) B

1000

1.0

M) M) M)

  

( ( ( 800 0.8

0.6

heme heme heme 600

0.4

Absorbance (AU) (AU) Absorbance Absorbance Absorbance (AU) Absorbance 0.2 400 0 450 500 550 600 650 700

200 Wavelength (nm)

ConcentrationConcentrationConcentration

0 0 10 20 30 40 50 Time (hours) Figure 2.9: A: Pharmacokinetics of L-PolyHb; B: Pharmacokinetics of H-PolyHb. Top – photograph of (1) bHb, (2) L- and H-PolyHb (prior to transfusion), (3) plasma (prior to transfusion), (4) end of transfusion, (5) 0.25 hour, (6) 0.5 hour, (7) 1 hour, (8) 2 hour, (9) 4 hour, (10) 8 hour, (11) 12 hour, (12) 24 hour and (13) 48 hour. Insets show representative visible spectra of plasma from the end of transfusion until 48 hours from 450 to 700 nm. The concentration (heme) versus time (hours) plot shows the plasma concentration ± sem (n=4/time point) of L- and H-PolyHb as total heme (black), ferrous heme (red) and ferric heme (blue).

45 Plasma heme concentrations versus time for L- and H-PolyHbs following 20% ET are shown in Figure 2.9. The pharmacokinetic parameter estimates for total PolyHb, ferrous PolyHb and ferric PolyHb following a 20% L- and H-PolyHb ET are listed in

Table 2.3. Doses received by animals (n=4/group) determined at the completion of ET were similar at 506.4 ± 0.42 mg (L-PolyHb) and 508.5 ± 0.77 mg (H-PolyHb).

Transfusions resulted in total plasma PolyHb maximum plasma concentrations (Cmax) of

14.4 ± 0.44 mg/mL and 13.1 ± 0.50 mg/mL for L- and H-PolyHb preparations, respectively. The volume of distribution (Vdss (mL)) was determined to be low (42.98 ±

5.5 (L-PolyHb) and 44.3 ± 0.67 (H-PolyHb)) suggesting a limited distribution from the central compartment for both preparations. Data plotted in Figure 2.9 and inset photos of representative plasma sets from L- and H- PolyHb dosed guinea pigs indicate visual differences in rates of circulatory clearance between the two preparations.

Pharmacokinetic parameters calculated from plotted data demonstrate a significantly increased rate of total circulatory clearance (Cl (mL•h-1)) of approximately 2-fold and decreased exposure expressed as area under the concentration versus time curve (AUC0-

-1 tlast and AUC0-∞ (mg•h•mL )) of approximately 2-fold following administration of H-

PolyHb compared to L-PolyHb. The circulating half-life (t1/2) of L-PolyHb was increased by approximately 1.5-fold compared to H-PolyHb.

In vivo generation of ferric (Fe3+) from ferrous (Fe2+) H- and L-PolyHb solutions

3+ demonstrated unique differences. The Cmax of ferric (Fe ) H-PolyHb was 4.57 ± 0.31

3+ mg/mL occurring at a Tmax of 4.0 ± 2.0 hours, while the Cmax of ferric (Fe ) L-PolyHb was 3.27 ± 0.32 at a Tmax of 8.0 ± 2.3 hours. Areas under the plasma concentration versus time curves were significantly greater for L-PolyHb. This finding is a function of the

46 overall greater circulation time of L-PolyHb, since the area under the ferric (Fe3+) L- and

H-PolyHb concentration versus time curves were determined to be 40% of their respective total PolyHb exposures.

Table 2.3: Pharmacokinetic parameter estimates following L- and H-PolyHb transfusion.

Estimate Total PolyHb PolyHb (Fe2+) PolyHb (Fe3+) L-PolyHb Dose (mg) 506.4 ± 0.42 489.1 ± 0.36 1.3 ± 0.10 -1 Cmax (mg•mL ) 14.4 ± 0.44 12.0 ± 0.43 3.27 ± 0.32 Tmax (h) 0.17 0.17 8.0 ± 2.3 90.1 ± 5.78 (41.9% AUC (mg•h•mL-1) 214.9 ± 9.91 124.7 ± 6.52 0-tlast total) 113.0 ± 20.9 (44.3% AUC (mg•h•mL-1) 252.4 ± 34.1 139.4 ± 14.5 0-∞ total) Cl (mL•h-1) 2.1 ± 0.24 3.6 ± 0.36 Vdss (mL) 42.98 ± 5.5 61.3 ± 7.2 t1/2 (h) 11.1 ± 0.22 6.85 ± 0.07 MRTi.v.(h) 22.15 ± 5.9 17.91 ± 4.0 -1 -3 -5 In vitro kox (min ) 1.23•10 ± 3.37•10

H-PolyHb Dose (mg) 508.5 ± 0.77 491.1 ± 1.0 1.0 ± 0.03 -1 † Cmax (mg•mL ) 13.1 ± 0.50 10.4 ± 0.70 4.57 ± 0.31 † Tmax (h) 0.17 0.17 4.0 ± 2.0 AUC (mg•h•mL-1) 51.9 ± 5.71† (40.5% 0-tlast 127.4 ± 11.2† 75.4 ± 12.1† total) AUC (mg•h•mL-1) 63.7 ± 8.3† (41.5% 0-∞ 158.5 ± 25† 94.8 ± 24.1† total) Cl (mL•h-1) 3.8 ± 0.40† 7.0 ± 1.2† Vdss (mL) 44.3 ± 0.67 69.4 ± 1.0 † t1/2 (h) 7.31 ± 0.53 5.60 ± 1.3 † MRTi.v.(h) 13.6 ± 1.9 13.3 ± 3.2 -1 -4 -5* In vitro kox (min ) 9.0•10 ± 3.34•10

† = Significant difference between L- and H-PolyHb parameter estimates (p<0.05) * = Significant difference between L- and H-PolyHb in vitro oxidation (p<0.05)

47 2.3.9 Plasma PolyHb Polymer Dissociation.

2.8 A 2.4

2.0

1.6

1.2

0.8

0.4

0.0 2.8

2.4 B Absorbance(405 units nm) Absorbance(405 units nm) 2.0

1.6

1.2

0.8

0.4

0.0 0 5 10 15 20 25 30 35 40 Minutes Figure 2.10: Circulating L- and H-PolyHb polymer distribution over time: (A) SEC of (····) bHb, (—) L- PolyHb and plasma samples from the end of transfusion to 48 hours (shown as dark grey fading to white). (B) SEC of (····) bHb, (—) H-PolyHb and plasma samples from the end of transfusion to 24 hours (shown as dark grey fading to white).

The plasma polymer distribution of L- and H-PolyHb over time is shown in Figure

2.10. Plasma samples from representative L- and H-state PolyHb transfused guinea pigs were analyzed on an analytical BioSep-S3000 size exclusion chromatography column and compared to elution profiles of PolyHb and bHb prior to infusion. Both L- and H-

PolyHb demonstrate similar polymer elution patterns over the time following the end of transfusion indicating limited hydrolysis of stabilized glutaraldehyde bonds in the L- and

H-PolyHb within the systemic circulation.

48 2.3.10 Oxidation of L- and H-PolyHb.

Figure 2.11: (A) In vitro autoxidation studies show a significant (p<0.05) attenuation in % H-PolyHb ferric Hb formation compared to % bHb () and % L-PolyHb (†) ferric Hb formation over 24 hours at 37°C. (B) % ferric Hb in plasma from in vivo studies show a significant (p<0.05) increase in % ferric H-PolyHb compared to L-PolyHb (†) within an early time frame following transfusion.

The differences in autoxidation rates obtained at 37°C and 24 hours were derived from a single-exponential, two parameter decay and are shown in Table 2.3 as the slope of the second parameter. The percent of ferric (Fe3+) formation at 2, 4 and 24 hours from in vitro and in vivo studies of oxidation are shown in Figure 2.11. The high O2 affinity 49 H-PolyHb solution demonstrates a significantly reduced rate of in vitro autoxidation and significantly less ferric Hb formation (H-PolyHb 2 hr: 8.23 ± 0.80%, 4 hr: 13.6 ± 1.03%) compared to bHb (2 hr: 13.7 ± 0.21%, 4 hr: 21.8 ± 0.44%) and low O2 affinity L-PolyHb

(2 hr: 32.8 ± 0.62%, 4 hr: 46.2 ± 0.78%) in the initial hours and at the 24 hour time point of autoxidation (bHb: 64.7 ± 1.36%, L-PolyHb: 79.2 ± 7.59%, H-PolyHb: 35.4 ± 2.0%).

A clear disconnect between in vitro autoxidation and in vivo oxidation is seen in Figure

2.11 A (in vitro autoxidation) and B (in vivo oxidation), where in vivo H-PolyHb is oxidized more rapidly to its ferric (Fe3+) form compared to L-PolyHb. In a dynamic in vivo situation involving both oxidative and clearance processes, H-PolyHb accumulated

3+ to 50.4 ± 1.4 % at a Tmax of 4 hours. This was significantly greater than ferric (Fe ) L-

PolyHb accumulation of 37.8 ± 2.8 % at a Tmax occurring 8 hours post transfusion.

2.4 Discussion

2.4.1 Biophysical Properties of L- and H-PolyHb Solutions.

The goal of this study was to synthesize high MW L- and H-PolyHbs with no tetrameric Hb and large molecular sizes (>500 kDa). bHb was polymerized in distinct quaternary states by carefully controlling the pO2 of the solution during the polymerization process. In addition, we characterized certain biophysical as well pharmacokinetic properties of the PolyHb solutions.

For L-PolyHb, the bHb solution was thoroughly deoxygenated before polymerization and polymerized under an inert argon atmosphere. The PolyHb obtained in this manner was constrained in the low O2 affinity quaternary state via intra- and inter-molecular glutaraldehyde cross-links. To produce H-PolyHb, bHb was first transformed into the

50 high O2 affinity R-state by completely oxygenating the bHb solution with O2 and subsequently polymerizing the bHb under an O2 saturated environment to ensure that the

H-PolyHb was constrained in the high O2 affinity quaternary state. After fractionating the

PolyHb mixture with a 500 kDa TFF cartridge, both L- and H-PolyHb solutions contained no tetrameric Hb. To our knowledge, this is the first time that L- and H-PolyHb solutions without Hb tetramers present were synthesized. The two commercially manufactured PolyHb solutions, HBOC-201® and PolyHeme, have made no mention of the pO2 during the Hb polymerization process [43, 140, 141]. Therefore, these commercial products may be considered heterogeneous with respect to the composition of the PolyHb quaternary state. Therefore, our high MW L- and H-PolyHbs can provide a better model for clinical evaluation.

The MW distribution of H-PolyHb is slightly higher than that of L-PolyHb even though the polymerization reaction was conducted at a lower G:Hb molar ratio. There are two reasons why this makes sense. First, it has been reported that the reactivity of glutaraldehyde to oxy-Hb is much greater than it is to deoxy-Hb [175, 176]. Thus, R-state polymerization can generate larger aggregates of bHb compared to T-state polymerization. Our results are consistent with that in the literature with respect to this phenomenon. The second reason is due to the presence of Na2S2O4 in the bHb solution.

Na2S2O4 was used in the T-state polymerization process to scavenge O2 from the bHb solution, and therefore maintain the pO2 at 0 mm Hg. Na2S2O4 can react with free aldehyde groups [225], thereby quenching some of the glutaraldehyde and reducing the actual G:Hb molar ratio to a level lower than the reported level of 50:1, hence, reducing the MW of L-PolyHb compared to H-PolyHb.

51 Despite the difference in the MW distribution of our L- and H-PolyHbs, both of them possess large MWs ranging from 16.59 to 26.33 MDa and no tetrameric Hb in solution

(Figure 2.3). Tetrameric Hb was removed from the PolyHb solution by filtering it through a 500 kDa TFF cartridge. The high MW of the PolyHb solutions and the absence of tetrameric Hb are important for several reasons. First, the tetrameric component of these PolyHb solutions is able to extravasate through the pores in blood vessels or interact more closely with endothelial cells covering the vascular lumen and can scavenge

NO from the surrounding endothelial cells or the sub-endothelial compartments. This will cause the smooth muscle cells to constrict leading to vasoconstriction in the microcirculation and eventual systemic hypertension [108]. These side-effects can be aggravated in a dose-dependent manner. However, it has been found that transfusion of

PolyHb solutions containing <1% of tetramic Hb caused no vasoconstriction [187, 208,

226]. The L- and H-PolyHbs synthesized in this study possess no tetrameric Hb and perhaps more importantly exceed 500 kDa in MW. Thus, it is reasonable to expect the L- and H-PolyHb solutions synthesized in this project should have less or no vasoactivity.

Secondly, high MW PolyHbs may exhibit longer circulation times compared to smaller

MW PolyHbs. It has been shown that the half-life of PolyHbs is proportional to the MW of the PolyHb [43, 227] and reached about 12-15 h for the 192 kDa fraction of

Hemolink® (Hemosol Corp., Mississauga, Canada) [227] to 20 h for the 576 kDa fraction of HBOC-201 [141]. Both of our PolyHbs possess MWs 50-fold greater than the 576 kDa fraction of HBOC-201. Thus, it is reasonable to predict that our L- and H-PolyHbs should display longer circulation times when dosed at equal concentration and volume.

52 After curve fitting the O2-PolyHb equilibrium data to the Hill equation, the P50 of both fractionated samples varied greatly (Figure 2.5). The P50 of the high MW L-PolyHb was around 41 mm Hg similar to the reported value of 38 mm Hg for HBOC-201® [21,

141], suggesting that the 50:1 L-PolyHb may have similar O2 transporting ability to

® HBOC-201 . The P50 of H-PolyHb is 0.66 mm Hg which demonstrates that cross- linking Hb in the R-state can greatly increase the O2 affinity of the product in a polymerization-dependent manner [175, 176]. Restitution of the O2 carrying capacity of low P50 materials compared to normal blood does not affect the total amount of O2 transported, but affects the amount of O2 released at each segment of the circulation.

Therefore, for the purpose of transfusion medicine, a moderate increase in O2 carrying capacity with decreased O2 affinity HBOCs should provide more effective O2 delivery.

Furthermore, a mixture of HBOCs with different P50s may provide an even more efficient mechanism for restoring optimal O2 delivery with a minimal amount of material.

The cooperativity of the two PolyHb solutions is <1 (Figure 2.5) compared to the reported value for unmodified bHb (~2.6) [209]. This is due to the intra- and intermolecular glutaraldehyde cross-links, which freezes the quaternary structure of Hb and reduces its structural flexibility. Therefore, the quaternary structure changes which otherwise would occur during normal O2 binding/offloading are hindered by the cross- links. This results in a significant loss of cooperative O2 binding to the Hb tetramer.

The metHb level of both PolyHbs were lower than 4% (Figure 2.6), which fulfill the standard 10% metHb requirement that is frequently cited in the literature [88]. Several steps were taken to retard autoxidation. First, the initially purified Hb had a metHb level lower than 1%. Second, the processes for deoxygenating and oxygenating bHb as well as

53 fractionation of the PolyHb mixture by TFF were all conducted in an ice bath in order to slow down oxidization of the heme. Third, PolyHb solutions were buffer exchanged against modified lactated Ringer’s solution containing N-acetyl-L-cysteine, an antioxidant [228], thereby limiting heme oxidation.

Stopped-flow kinetic analysis revealed some interesting changes in the gaseous ligand binding properties which resulted from the polymerization of Hb with the cross- linking reagent glutaraldehyde. Although dioxygenation of NO by oxygenated Hb is usually not sensitive to Hb conformational changes or chemical modifications, the kinetic parameters describing O2 dissociation and CO binding are good indicators of Hb O2 affinity. Not surprisingly, the differences found in O2 dissociation (koff, O2) and CO binding (kon, CO) of L- and H-PolyHb with unmodified bHb are consistent with their equilibrium O2 binding properties (i.e. O2 affinity (P50)). Moreover, the large drop in the

P50 of H-PolyHb compared to bHb is mostly reflected by its 20-fold increase in the CO binding rate constant, suggesting a much more open conformation assumed by the H-

PolyHb that leads to higher heme pocket accessibility. A previous study examined the fast kinetics of the glutaraldehyde polymerized bHb veterinary product Oxyglobin

(Biopure Corp., Cambridge, MA) and its 4 component fractions (F1(multi-tetramer)-F4

(single tetramer)) ranging in molecular size from tetrameric (~64 kDa) to multi-tetrameric

(~500 kDa) [229]. The starting material (bHb) in this study exhibited a similar koff, O2

-1 -1 -1 (33.5 ± 0.1 s ) and kon, CO (0.22 ± 0.02 M s ) to the bHb used in the present study (see

 -1 -1 Table 2.2). koff, O2 values for Oxyglobin (60.0 ± 3.2 s ) and fractions (F1, 63.3 ± 1.2 s ,

F2, 57.2 ± 1.0 s-1, F3, 63.7 ± 1.4 s-1, and F4, 62.8 ± 2.5 s-1) were nearly identical to the L-

 PolyHb described in the current study. Similarly, kon, CO values for Oxyglobin (0.15 ± 54 0.01 M-1 s-1) and fractions (F1, 0.19 ± 0.01 M-1 s-1, F2, 0.18 ± 0.01 M-1 s-1, F3, 0.20 ±

0.02 M-1 s-1, and F4, 0.19 ± 0.02 M-1 s-1) were nearly similar to the L-PolyHb described in the current study (see Table 2.2) [229].

At PolyHb concentrations of 5 and 10 g/dL, the viscosity of both PolyHb solutions is higher than that of blood (~ 3 cp). In this study, the viscosity of both PolyHb solutions increases as the PolyHb concentration increases. This should come as no surprise, since molecular interactions between PolyHb molecules will be enhanced as the concentration increases in solution. Originally RBC substitutes were designed with the assumption that lower blood viscosity is always beneficial. However, blood viscosity directly influences blood vessel diameter due to the shear stress interaction with the endothelium [230]. It is known that a decrease in blood viscosity induces vasoconstriction. Hence, blood viscosity is an important determinant of vasoactivity, as shown in our experiments [231]. PolyHbs can interact mechanically in terms of shear stress, presumably leading to a difference in mechanotransduction with the endothelium. Transfusion of these high viscosity solutions may be advantageous, since these solutions could stimulate NO generation via mechanotransduction of the endothelium [203]. The release of NO would relax the tone of blood vessels and alleviate the vasoconstrictive effect. Additionally, as the PolyHb solutions are proposed to be used during anemic conditions, where blood viscosity is already low, the increase in plasma viscosity induced by the PolyHb will not increase peripheral vascular resistance.

Both polymers showed very similar effects on RBC aggregation. Although the degree of RBC aggregation was substantially less when compared to a high viscosity plasma expander (6% dextran 500 kDa). The ability of the larger bHb polymers to 55 promote RBC aggregation was microscopically evident, and the aggregates tended to form rounded and compressed clumps rather than elongated rouleaux.

Interestingly, the COP of both PolyHbs solutions is low (< 10 mm Hg) (Table 2.2).

This is due to two reasons. First, there is no free Hb present in the PolyHb solution [43].

Second, filtering the PolyHb solutions through the 500 kDa TFF column substantially removed Hb species smaller than 500 kDa. The COP of the PolyHb solutions is lower than that of normal blood (27 mm Hg), and can be adjusted to physiological levels by supplementing PolyHb solutions with human serum albumin solution. When transfused, this should enable simultaneous O2 transport and blood volume expansion. Moreover, O2 carrying capacity is in principle a direct function of the concentration of functional Hb molecules. The present PolyHb formulations have the highest O2 carrying capacity due to their extremely low COP. Therefore, these solutions will not elicit autotransfusion and dilute the HBOC concentration in the blood, since their COP is lower than that of blood.

As for other commercial HBOCs, augmenting their concentration is not an option in order to increase their O2 carrying capacity, since they have increased COP. The high

COP will promote the flow of interstitial fluid into the circulation, diluting the HBOC, thus lowering the circulating concentration of Hb and increasing the blood volume, a self- limiting process. In contrast, the PolyHbs described in this work constitute a novel set of

HBOCs that could potentially overcome these problems.

2.4.2 Pharmacokinetics of L- and H- PolyHb Solutions.

Pharmacokinetic evaluation of both L- and H-PolyHbs revealed an extended circulation time for L- versus H-PolyHb. All pharmacokinetic estimates of clearance and

56 exposure, excluding Cmax which was intentionally matched, indicated that H-PolyHb was removed from the circulation at a significantly greater rate than L-PolyHb. This finding is somewhat contrary to the expected result that increased molecular size (H-PolyHb ~ 26

MDa versus L-PolyHb ~ 17 MDa) contributes to a greater circulation time. Interestingly, while behaving as expected from the perspective of in vitro autoxidation, H-PolyHb (high

O2 affinity) is oxidized in vivo to a greater extent than L-PolyHb (low O2 affinity). The observation of increased oxidation of H-PolyHb within an earlier time frame following transfusion may have contributed to the greater overall clearance of this protein in circulation.

2.5 CONCLUSION:

In this work, ultrahigh MW low O2 affinity L-PolyHb (MW = 16.59 MDa and P50 =

41 mm Hg) and high O2 affinity H-PolyHb (MW = 26.33 MDa and P50 = 0.66 mm Hg) were synthesized with no tetrameric Hb and low metHb levels (<4%). Both PolyHbs possessed high viscosities and low COPs. In addition, transfusion of these PolyHbs indicated limited PolyHb dissociation, which is a good preliminary indicator that these

PolyHb will not extravasate through blood vessels and scavenge NO. In light of these results, these PolyHbs should not elicit vasoconstriction/hypertension and are a good basis for future HBOC development.

57 CHAPTER 3

The Effect of Cross-link Density on the Biophysical Properties and Oxygenation

Potential of PolyHbs with Low and High O2 Affinity

3.1 Introduction

In the previous chapter, ultrahigh MW glutaraldehyde PolyHbs with distinctively different O2 affinities, absence of tetrameric Hb and low metHb levels were synthesized and characterized both in vitro and in vivo. These PolyHbs displayed very little to no vasoactivity and limited polymer dissociation in vivo [132, 190]. These results indicate that polymerization of Hb to yield high MW polymers free of tetrameric Hb and low MW polymeric species can be an effective approach to produce a safe and effective HBOC.

However, the use of these ultrahigh MW PolyHbs may be hampered by some intrinsic drawbacks. One particular study showed that the >500 kDa fraction of 50:1 L-

PolyHb can cause moderate hypertension at high doses mainly due to its high solution viscosity [190]. Thus, more work should be done to further investigate the effect of glutaraldehyde based polymerization on the biophysical properties of PolyHb solutions.

Previously, a small library of PolyHb solutions was synthesized with glutaraldehyde to

Hb (G:Hb) molar ratios ranging from 10:1 to 50:1 [130] in order to evaluate the effect of varying the G:Hb molar ratio on the physical properties of PolyHbs [130]. However, the

PolyHbs obtained in these studies were synthesized in a heterogeneous quaternary state

58 [130]. Therefore in this chapter, a systematic investigation will be conducted to probe the effects of varying the G:Hb molar ratio on the biophysical properties of PolyHbs polymerized in distinctively either the low (L-PolyHb) or high (H-PolyHb) O2 affinity state in order to optimize the synthesis of PolyHb for various biomedical applications.

Purified bHb will be polymerized with G:Hb molar ratios ranging from 10:1 to 30:1 and will be diafiltrated against a modified lactated Ringer’s buffer to remove any remaining glutaraldehyde, NaCNBH3 and NaBH4 as well as to obtain the MW fraction >

500 kDa. The resulting PolyHb solutions will then be characterized to investigate the effect of varying the cross-link density on their biophysical properties.

In addition, a hollow fiber (HF) bioreactor model will be used to assess the ability of

PolyHbs to transport O2 from the lumen of individual HFs to surrounding cultured cells.

A typical HF bioreactor is composed of a closed cylindrical cartridge used to house the cells and a bundle consisting of hundreds to thousands of porous cylindrical HF membranes fixed between the two ends of the cartridge for nutrient and waste transport

[232]. The continuously recirculating flow of medium in the HF supplies O2 and nutrients to cultured cells and carries away metabolic wastes, which is a good mimic of the structure of blood vessels [232]. We choose this approach, since in vivo evaluation of this small library of materials would be cost-prohibitive and would eventually yield a more focused selection of PolyHb candidates for future in vivo studies.

59 3.2 Materials and Methods

3.2.1 Materials

Glutaraldehyde (70%), NaCl, KCl, NaOH, Na2S2O4, NaCl, KCl, CaCl2-2H2O, NaOH, sodium lactate, N-acetyl-L-cysteine, NaCNBH3 and NaBH4 were purchased from Sigma-

Aldrich (Atlanta, GA). 100 kDa and 500 kDa HF cartridges were purchased from

Spectrum Labs (Rancho Dominguez, CA). KCN, K3Fe(CN)6 and all other chemicals were purchased from Fisher Scientific (Pittsburgh, PA).

3.2.2 Hb Purification.

bHb was purified from lysed bovine RBCs by TFF as described in the literature [209,

210, 233].

3.2.3 Polymerization of bHb.

L- and H-PolyHb solutions were synthesized according to the procedures outlined in chapter 2 at the following G:Hb molar ratios: 10:1, 20:1 and 30:1. All reactions were repeated in triplicate.

3.2.4 Clarification and Diafiltration of PolyHb Solution.

The resulting PolyHb solutions were initially clarified by filtering them through a column packed with autoclaved glass wool [212] to remove large particles. The clarified

PolyHb solution was then diluted from 1500 mL to 2000 mL and then subjected to 4 cycles of diafiltration via TFF against an ice-cold modified lactated Ringer’s solution

(NaCl 115 mmol/L, KCl 4 mmol/L, CaCl2-2H2O 1.4 mmol/L, NaOH 13 mmol/L, sodium

60 lactate 27 mmol/L and N-acetyl-L-cysteine 2 g/L). 100 kDa HF cartridges were used for diafiltration of both L- and H-PolyHb solutions with a G:Hb molar ratio of 10:1, while

500 kDa HF cartridges were employed for PolyHb solutions with G:Hb molar ratios of

20:1 and 30:1. For each diafiltration cycle, the PolyHb solution was concentrated to a volume less than 200 mL. At the final diafiltration step, the resulting concentrated

PolyHb solution was immediately stored at -80oC for future use.

3.2.5 MetHb Level and Protein Concentration of PolyHb Solutions.

The metHb level of bHb/PolyHb solutions was measured via the cyanomethemoglobin method [234]. Total protein concentration was measured according to the Bradford method [215] using the Coomassie Plus protein assay kit (Pierce

Biotechnology, Rockford, IL).

3.2.6 Equilibria of O2-bHb/PolyHb Solutions.

O2-bHb/PolyHb equilibrium curves were measured using a Hemox Analyzer (TCS

Scientific Corp., Southampton, PA) at 37oC. Samples were diluted with 5 mL of Hemox buffer (pH 7.4, TCS Scientific Corp.), 20 μL of Additive A, 10 μL of Additive B and 10

μL of antifoaming agent to a protein concentration of 30 M (heme) followed by

o equilibration with compressed air to a pO2 of 145±2 mm Hg at 37 C. Nitrogen was then used to deoxygenate the sample solution while the bHb/PolyHb O2 saturation (Y) was measured as a function of pO2. The P50 (pO2 at which the bHb/PolyHb is half-saturated with O2) and cooperativity coefficient (n) were regressed from curve fits of the

61 experimental O2-bHb/PolyHb equilibrium curves to the Hill equation using IGOR Pro

(WaveMetrics Inc, Lake Oswego, OR) [216].

3.2.7 SDS-PAGE and Native-PAGE of PolyHb Solutions.

The MW distribution of bHb/PolyHb solutions was initially assessed via SDS-PAGE and native-PAGE using a Mini-PROTEAN 3 Cell (Bio-Rad; Hercules, CA). A 4% stacking gel with a 12% resolving gel was employed for SDS-PAGE, while a 7.5% precast native gel (Bio-Rad) was employed for Native-PAGE. Each lane was loaded with approximately 25 µg of protein. The gel was run at 120 V for approximately 1 hour.

After electrophoresis, the gel was stained with Coomassie blue R250 (stain buffer, Bio-

Rad) overnight, and then destained with a buffer consisting of 10% acetic acid and 20% methanol. The gel was scanned on a Gel Doc XR (Bio-Rad) imaging system for further analysis.

3.2.8 Absolute MW Distribution of bHb/PolyHb Solutions.

The absolute MW distribution of bHb/PolyHb solutions was characterized by size exclusion chromatography coupled with multi-angle static light scattering (SEC-MASLS) as described previously in the literature [132].

3.2.9 Viscosity and Colloid Osmotic Pressure of bHb/PolyHb Solutions.

The viscosity of bHb/PolyHb solutions was measured in a cone and plate viscometer

DV-II plus with a cone spindle CPE-40 (Brookfield Engineering Laboratories,

62 Middleboro, MA), at a shear rate of 160/sec. In contrast, the colloid osmotic pressure

(COP) of bHb/PolyHb solutions was measured using a Wescor 4420 Colloid Osmometer

(Wescor, Logan, UT) [218].

3.2.10 Stopped Flow Kinetic Analysis of bHb/PolyHb Solutions.

The rapid kinetics of gaseous ligand reactions with bHb/PolyHb was measured in an

Applied Photophysics SF-17 micro-volume stopped-flow apparatus as previously described in the literature [217]. bHb/PolyHb solutions (30 M in heme) were rapidly mixed with equal volumes of 1.5 mg/mL sodium dithionite (British Drug House, Poole,

England), and O2 dissociation was monitored by the absobance changes at 437.5 nm in

0.05 M bis-Tris buffer, pH 7.4. Multiple kinetic traces were averaged for each reaction, and fit to exponential equations using Marquardt-Levenberg fitting routines in the

Applied Photophysics software. The kinetics of CO binding with deoxygenated bHb/PolyHbs was measured at 437.5 nm in 50 mM bis-Tris buffer at pH 7.4 at 25C in the presence of sodium dithionite. The CO stock solution (about 1 mM) was prepared by saturating the degassed buffer solution with the flow of pre-washed CO gas.

The kinetics of NO oxidation with oxy-bHb/PolyHb solutions was measured in the stopped-flow instrument as previously described [131]. NO stock solutions (~ 2 mM) were prepared by saturating deoxygenated 0.05 M Tris buffer, pH 7.4, in a gas-tight serum bottle with NO gas that was pre-washed with deoxygenated 1 M NaOH and buffer

63 solutions. The NO stock solution was then transferred with a Hamilton syringe to a gastight syringe containing deoxygenated buffer solution to make appropriate concentrations of NO solutions. bHb/PolyHb solutions (1 M in heme) were mixed with

NO solutions ( 50 μM), and the absorbance changes of the reaction were followed at

420 nm. Multiple traces were averaged for each reaction, and fit to exponential equations to obtain apparent reaction rate constants.

The oxygen binding rate constant (kon, O2) based on a per heme basis is derived from the reaction scheme shown in equation 3.1 and makes use of equations 3.2~3.4, which

results in equation 3.5. Therefore (kon, O2) can be calculated using measured equilibrium and kinetic parameters.

kon, O2 O2 +HBOC oxyHBOC (3.1) koff, O2

RoxyHBOC =- R O2 =kkon , O 2 *[O2]*[HBOC]- off , O 2 *[oxyHBOC] (3.2)

[HBOC]total =[HBOC]+[oxyHBOC] (3.3)

n [oxyHBOC] [HBOC] pO2 eq S= , 1-S= , S = nn [HBOC]total [HBOC] total P 50 pO 2 (3.4)

kkoff, O22*S*S eq off , O eq RoxyHBOC = 0 at equilibriumkon, O2   (3.5) [O2 ]*(1-S eq ) *pO 2 *(1-S eq )

-3 3 Where  is the solubility of O2 in aqueous media, which is 1.71*10 mol/(m -mm Hg)

[235].

64 3.2.11 Hydrodynamic Molecular Size and Zeta Potential of bHb/PolyHb

The hydrodynamic molecular diameter and zeta (ζ) potential of bHb and PolyHb solutions were measured on a Zetasizer Nano-ZS system (Malvern Instruments Ltd,

Worcestershire, UK) at 37oC. bHb/PolyHb solutions were first diluted with PBS (pH 7.4), which had been previously filtered through a 0.22 μm membrane, to approximately 2 mg/mL followed by equilibration at 37oC. Dynamic light scattering was used to determine molecular size, while Doppler velocimetry and phase analysis light scattering

(PALS) was used to measure the ζ potential.

3.2.12 In Vitro Autoxidation of PolyHb Solutions

All PolyHb samples were converted to the oxy-PolyHb form and diluted to A540

0.8~0.9 with air equilibrated 50 mM Chelex-treated potassium phosphate buffer prior to measurements. Experiments were conducted in sealed cuvettes at 37°C with bHb as the control. Absorbance changes in the range of 450-700 nm were recorded over 40 hours due to the spontaneous oxidation of Hb in a temperature-controlled Cary 100 UV-Visible

Spectrophotometer (Agilent Technologies, Inc, Santa Clara CA). Autoxidation rate constants were calculated from nonlinear least square curve fitting of the % oxyHb time course via Sigma Plot (Systat Software Inc. San Jose, CA). All experiments were conducted in triplicate.

65 3.2.13 Simulation of bHb/PolyHb Facilitated O2 Transport in a Hepatic HF

Bioreactor.

The O2 transport model is based on the geometry of a single HF contained as a bundle in a HF bioreactor which mimics the in vivo capillary/sinusoid structure (Figure

3.1). In this geometry, cell culture media containing bHb/PolyHb solution flows through the lumen of the HF bioreactor to provide nutrients to cells (in this case hepatocytes) which reside in the extra capillary space (ECS), while simultaneously carrying away metabolic waste products. The cell culture media recirculates throughout the entire HF bioreactor system. The HF membrane has a MW cut-off of 35 kDa and therefore confines bHb/PolyHbs (MW > 64 kDa) within the lumenal space of the HF without directly contacting cells cultured within the ECS.

The velocity profile in each of the three subdomains (lumen, membrane, and ECS) is calculated from a set of momentum transport partial differential equations (PDEs), shown in equation 3.6.

    2 Navier-Stokes Equation (lumen): v'''' v     P     v (3.6.A) l0 v 0   l 0 v 0 

2 l0 2 Brinkman’s Equation (membrane and ECS): v'''   P   v (3.6.B) 

vP Where vP',' . v ' is the dimensionless velocity vector and P ' is the v0 v 0/ l 0 dimensionless pressure. l0, v0,  and  represent the reference length, reference velocity, fluid density and viscosity, respectively.

66 The mass conservation equations which describe transport of dissolved O2, total

HBOC and O2-HBOC in dimensionless form are shown in equation 3.7. C can either represent the dimensionless O2 partial pressure (pO2), total HBOC concentration or O2-

HBOC concentration. C0 can either represent the reference O2 partial pressure, total

HBOC concentration or O2-HBOC concentration. D can either represent the diffusivity of

O2 or HBOC. R represents the rate of formation of O2/O2-HBOC. The HBOC diffusivity was estimated from the weight-averaged MW listed in Table 3.1 using equation 3.8.

2 l0R  v 0 l 0  C'''    v  C (3.7) DCD0 

D1.013 104 ( MW ) 0.46 cm 2 / s (3.8)

The reaction between O2 and HBOC is described by equation 3.9 [236], where m is the number of O2 binding sites on a single HBOC molecule. Given the thermodynamic relationship describing the equilibrium between HBOC and O2 (equation 3.10, where a1- a4 are the Adair constants), the rate of formation of O2 ( R (mm Hg/s)) or O2-HBOC O2

3 (RoxyHBOC (mol/m *s)) in the lumen is shown in equation 3.11. S is defined as the HBOC saturation, i.e. the molar fraction of HBOC that is saturated with O2 and Seq is the HBOC saturation fraction at equilibrium. [O2] is the concentration of dissolved O2, [HBOC] is the concentration of total HBOC and  is the solubility of O2 in aqueous media. The O2 consumption rate in the ECS is described by Michaelis-Menten (M-M) kinetics (3.12), where VM is the maximum O2 consumption rate of hepatocytes and kM is the Michaelis constant of hypatocytes.

67 k ' on, O2  mO2  HBOC oxyHBOC (3.9) k'off, O 2

a pO 2 a  pO2  3 a  pO 3  4 a  pO 4 S  1 2 2 2 3 2 4 2 (3.10) eq 2 3 4 41 a1  pO 2  a 2  pO 2  a 3  pO 2  a 4  pO 2 

 Seq R   R  k'off, O2  [ HBOC ] 1  S  S (3.11) oxyHBOC O2  mS1 eq

VM *2 pO RO2  (3.12) kM  pO2

Further details about the mathematical model and parameters/constants used in the simulations can be found in the literature [237]. The coupled set of PDEs was solved by the finite element method in Comsol Multiphysics (COMSOL, Inc., Burlington, MA) yielding numerical solutions.

Figure 3.1: Hepatic HF bioreactor system. A: Schematic of the entire bioreactor system. B: Expanded view of the HF bioreactor cartridge. C: Geometry of a single HF used in the computer simulations.

68 3.3 Results

bHb was polymerized using glutaraldehyde as the cross-linking reagent at a G:Hb molar ratio of 10:1, 20:1 and 30:1 for both L-PolyHb and H-PolyHb. After polymerization, each PolyHb mixture was fractionated and further diafiltrated using TFF against ice cold modified lactated Ringer’s buffer to remove tetrameric Hb and smaller

Hb oligomers. For both L- and H-PolyHb solutions with a G:Hb molar ratio of 10:1,

PolyHb fractions with MW above 100 kDa were used in this study while for L- and H-

PolyHb solutions with a G:Hb molar ratio of 20:1 and 30:1, PolyHb fractions with MW above 500 kDa were used in this study.

3.3.1 pO2 of bHb Solutions During the Polymerization Process.

By using the same pO2 level control strategy as mentioned in chapter 2, the pO2 of bHb solutions was maintained at 0 mm Hg at all stages of the polymerization process for the preparation of L-PolyHbs, while it remained above the measurement range of the blood gas analyzer during the polymerization of H-PolyHbs. Therefore, bHb was kept exclusively in either the fully deoxygenated or fully oxygenated state during the polymerization reaction. The pO2 of all PolyHbs solutions dropped to 0 mm Hg after quenching due to the reduction of dissolved O2 in solution by NaBH4.

69 3.3.2 Effect of Cross-link density on MW Distribution of PolyHb Solutions

Figure 3.2: SDS-PAGE of bHb/PolyHb solutions.

Figure 3.3: Native-PAGE of bHb/PolyHb solutions.

Figures 3.2 and 3.3 show the SDS-PAGE and native-PAGE of bHb, L- and H-

PolyHbs, respectively. All PolyHbs exhibited no bands corresponding to individual /

70 subunits in the SDS-PAGE (Figure 3.2). However, both 10:1 L- and H-PolyHbs showed faint bands close to 32 kDa (2/2/ dimers) and 64 kDa (tetrameric Hb) and weak bands above 70 kDa (Figure 3.2). Native-PAGE of 10:1 L- and H-PolyHbs corroborates the SDS-PAGE results and reveals bands corresponding to the bHb tetramer (Figure 3.3).

Interestingly, all PolyHbs with G:Hb molar ratios greater than 10:1 showed strong bands above 250 kDa, some diffuse smearing from 50 to 250 kDa and no bands corresponding to individual / subunits, 2/2/ dimers or tetramers (Figures 3.2 and 3.3). These results indicate that the degree of bHb polymerization increases with increasing G:Hb molar ratio.

Figure 3.4: Absolute MW distribution of bHb/PolyHb solutions.

71 Figure 3.4 shows the absolute MW distribution of both L- and H-PolyHbs.

Unmodified bHb was used as a control (MW  64 kDa). Light scattering results suggest the presence of free Hb in 10:1 L- and H-PolyHbs and the absence of free Hb in PolyHb solutions with higher G:Hb molar ratio.

The MW of all PolyHbs is listed in Table 3.1. The MW of PolyHbs increased with increasing G:Hb molar ratio, while H-PolyHbs possessed higher MW than that of the corresponding L-PolyHbs at the same G:Hb molar ratio, which is expected based on previous studies in the literature [130, 132, 175, 176].

Table 3.1: Biophysical properties of bHb/PolyHb solutions. The error bars represent the standard deviation from triplicate reactions.

Solution P50 (mm Hg) n MetHb Level (%) MW (kDa) bHb 27.37±1.57 2.84±0.081 0.47±0.099 65.3±0.6 10:1 L-PolyHb 29.66±0.82 1.36±0.079 3.59±1.20 91±10 20:1 L-PolyHb 37.10±0.94 1.05±0.098 1.95±0.24 749±43 30:1 L-PolyHb 41.16±3.05 1.01±0.015 2.90±1.45 1330.3±159 10:1 H-PolyHb 4.54±1.20 0.61±0.023 1.23±0.13 137.950±21.8 20:1 H-PolyHb 2.18±0.90 0.56±0.047 4.54±1.02 1025.9±122.8 30:1 H-PolyHb 1.84±0.78 0.69±0.11 4.50±0.98 6256±886.7

3.3.3 Effect of Cross-link Density on Hydrodynamic Diameter and Zeta Potential of

PolyHbs

The hydrodynamic molecular diameter and zeta (ζ) potential of L- and H-PolyHb solutions were measured and listed in Table 3.2 with unmodified bHb as the control. The hydrodynamic diameter of unmodified bHb measured at 37°C in PBS solution (pH 7.4) was 8 nm, which is in agreement to the reported value of 7 nm in the literature [238]. The

72 hydrodynamic diameter of PolyHbs was larger than that of bHb and increased with increasing MW independent of the O2 affinity. Similarly, the absolute value of the ζ potential of PolyHb solutions was higher than that of unmodified bHb. However, the ζ potential of PolyHb solutions didn’t strictly increase with the MW of the PolyHbs. In the subset of either L- or H-PolyHb, although PolyHbs with the highest G:Hb molar ratio

(30:1) possessed the highest ζ potentials, similar ζ potentials were observed for the

PolyHbs with G:Hb molar ratio of 10:1 and 20:1. In addition, the overall ζ potentials of

L-PolyHb solutions were slightly higher than that of H-PolyHb solution.

Table 3.2: Molecular diameter and zeta potential of bHb/PolyHb.

Hydrodynamic Solution ζ Potential (mV) Diameter (nm) bHb 8.0±0.02 -6.8±0.4

10:1 L-PolyHb 10.8±2 -11.5±1.7

20:1 L-PolyHb 23.8±5.5 -11.1±0.8

30:1 L-PolyHb 31.4±0.4 -14.3±0.4

10:1 H-PolyHb 13.0±2.3 -7.5±1.4

20:1 H-PolyHb 24.5±0.2 -6.9±0.7

30:1H-PolyHb 73.5±9.8 -10.1±0.4

3.3.4 Effect of Cross-link density on Oxygen Affinity and Cooperativity of PolyHb

Solutions.

Figure 3.5 shows the O2-bHb/PolyHb equilibrium curves of unmodified bHb, L- and

H-Poly Hbs. All PolyHbs exhibited hyperbolic-shaped equilibrium curves, which indicate 73 a substantial loss in cooperativity. The O2-L-PolyHb equilibrium curves are increasingly shifted to the right of the O2-bHb equilibrium curve with increasing G:Hb molar ratio. In contrast, the O2-H-PolyHb equilibrium curves are increasingly shifted to the left of the

O2-bHb equilibrium curve with increasing G:Hb molar ratio. The dependence of the regressed P50 and cooperativity coefficient (n) of PolyHb solutions on the G:Hb molar ratio is shown in Table 3.1 with unmodified bHb as the control. The P50s of L-PolyHbs are higher than that of unmodified bHb (27.36±1.57 mm Hg) and increase with increasing G:Hb molar ratio. In contrast, all H-PolyHbs display extremely low P50s compared to both unmodified bHb and L-PolyHbs, which decrease with increasing G:Hb molar ratio.

The cooperativity coefficients (n) of both L- and H-PolyHbs are lower than that of unmodified bHb (2.82±0.081). The n of L-PolyHbs decreased with increasing G:Hb molar ratio, while all H-PolyHbs in our study displayed similar cooperativity coefficients close to 0.6 in value, irrespective of the G:Hb molar ratio.

o Figure 3.5: O2-bHb/PolyHb equilibrium curves measured at 37 C. The dashed lines represent experimental data, while the solid lines represent curve fits to the Hill equation [216].

3.3.5 Effect of Cross-link Density on MetHb Level of PolyHb Solutions.

The metHb level of unmodified bHb was extremely low (approximately 0.5%), while

L- and H-PolyHb solutions exhibited metHb levels below 6% (Table 3.1).

3.3.6 Effect of Cross-link Density on Viscosity and COP of PolyHb Solutions

Table 3.3 shows the viscosity and COP of both L- and H-PolyHb solutions with unmodified bHb as the control. At a total protein concentration of 10 g/dL, the viscosity of both L- and H-PolyHb solutions are higher than that of unmodified bHb and increase with increasing G:Hb molar ratio. In contrast, the COP of PolyHb solutions decreased

75 with increasing G:Hb molar ratio. However, L-PolyHb solutions exhibited lower COPs compared to H-PolyHb solutions when compared at the same G:Hb molar ratio.

Table 3.3: Viscosity and COP of bHb/PolyHb solutions.

Concentration Viscosity COP Solution (g/dL) (cp) (mm Hg) bHb 10 1.6 38 10:1 L-PolyHb 10 3.2 42 10:1 H-PolyHb 10 2.7 48 20:1 L-PolyHb 10 4.8 24 20:1 H-PolyHb 10 3.6 39 30:1 L-PolyHb 10 14.8 2 30:1 H-PolyHb 10 9.8 14

3.3.7 Effect of Cross-link Density on Stopped Flow Kinetic Analysis of PolyHb

Solutions

The kinetic parameters for O2 dissociation (koff, O2), CO association (kon, CO) and NO dioxygenation (kox, NO) were measured for unmodified bHb, L- and H-PolyHbs (Table

3.4). However, the O2 association (kon, O2) rate constant was calculated using equation 5

- and is listed in Table 3.4. The O2 dissociation rate constant for unmodified bHb is 36.1 s

1, while that of PolyHb solutions deviate from that of unmodified bHb by a 30-60% increase for L-PolyHbs and a 17-40% reduction for H-PolyHbs, respectively. The O2 association rate constants of L-PolyHbs decrease with increasing G:Hb molar ratio, while those of H-PolyHbs increase with increasing G:Hb molar ratio, at pO2 levels of 80 and

150 mm Hg. The O2 association rate constant of unmodified bHb is the same order of magnitude as that of human Hb (5-40 M-1s-1) [239] and larger than that of any PolyHb.

76 The CO association rate constants for H-PolyHbs exhibited a substantial increase of over

20-fold compared to that of unmodified bHb. In contrast, L-PolyHbs displayed a slightly slower CO association rate compared to unmodified bHb, which decrease with increasing

G:Hb molar ratio. Interestingly, the NO dioxygenation rate constants remained largely unchanged among unmodified bHb, L- and H-PolyHbs.

Table 3.4: Kinetic parameters of bHb/PolyHb solutions.

-1 -1 -1 -1 -1 k (M s ) k (M s ) k k k (s ) on, O2 on, O2 on, CO ox, NO Solution off, O2 -1 -1 -1 -1 (pO2= 80 mm Hg) (pO2= 150 mm Hg) (M s ) (M s ) bHb 36.1 5.55 17.65 0.22 18.3 10:1 L-PolyHb 47.2 1.33 1.67 0.193 18.9 20:1 L-PolyHb 58.6 0.96 0.99 0.183 17.1 30:1 L-PolyHb 57.1 0.817 0.821 0.157 18.7 10:1 H-PolyHb 29.1 1.22 0.96 6.138 17.4 20:1 H-PolyHb 29.7 1.63 1.24 3.92 16.4 30:1 H-PolyHb 24.7 2.44 2.01 5.95 17.5

3.3.8 Autoxidation of PolyHb

Table 3.5: Autoxidation rate constant of bHb/PolyHb solutions

Autoxidation rate constant Solution (h-1) bHb 0.0212±0.00255

10:1 L-PolyHb 0.0486±0.00573

20:1 L-PolyHb 0.0537±0.0103

30:1 L-PolyHb 0.0557±0.0054

10:1 H-PolyHb 0.0342±0.0105

20:1 H-PolyHb 0.0335±0.00079

30:1 H-PolyHb 0.0333±0.00527

77 The autoxidation rate constant of PolyHb solutions and unmodified bHb are listed in

Table 3.5. PolyHb solutions demonstrated higher autoxidation rate constants compared to unmodified bHb. In the subset of L-PolyHbs, autoxidation rate constants increased with increasing cross-link density, while the autoxidation rate constant of H-PolyHb solutions was not sensitive to the cross-link density. Additionally, H-PolyHb solutions exhibited lower autoxidation rates compared to L-PolyHb solutions under similar experimental conditions.

3.3.9 Simulation of bHb/PolyHb Facilitated O2 Transport in a Hepatic HF

Bioreactor.

Figure 3.6: O2 consumption rate normalized by the control (no HBOC supplementation). A: represents L- PolyHb solutions. B: represents H-PolyHb solutions. The biophysical parameters used to simulate 50:1 L- and 40:1 H-PolyHb were taken from the literature [132].

An O2 transport model was used to simulate O2 transport within a single HF of a hepatic HF bioreactor, in which the circulating cell culture media was supplemented with

78 bHb/PolyHbs. The case where no HBOC was present in the cell culture media served as the control, and all results were normalized on this basis. The oxygenation potential of unmodified bHb and Oxyglobin® (a PolyHb manufactured for veterinary use, OPK

Biotech, Cambridge, MA) in the HF bioreactor was also simulated for comparison.

The normalized O2 consumption rate of C3A hepatocytes cultured in a HF bioreactor supplemented with bHb/PolyHb is shown in Figure 3.6 as a function of the inlet pO2

(pO2in). The computed value of the normalized O2 flux monotonically decreased as the pO2in increased for all HBOCs studied. At low pO2ins (< 40 mm Hg), the predicted O2 consumption rate with either L- or H-PolyHb supplementation was lower than that of unmodified bHb and Oxyglobin®, and decreased with increasing G:Hb molar ratio. In contrast, at higher pO2ins (~150 mm Hg), the computed O2 consumption rate for bHb supplemented HF bioreactors was similar to that with either L- or H-PolyHb supplementation at all G:Hb molar ratios.

Figure 3.7: pO2 profiles within a single HF (lumen, membrane and ECS) with varying heme concentration. 100% [Heme] = 15 g/dL of bHb. The biophysical parameters used to simulate 50:1 L- and 40:1 H-PolyHb were taken from the literature [132].

Figure 3.7 shows the simulated pO2 profiles within a single HF (including lumen, membrane and ECS) under varying concentrations of PolyHb. In this work, the heme concentration was normalized by the average physiological heme concentration in human blood (8800 M). Each rectangular unit in the figure represents a cross-sectional view of a single HF (Figure 3.1C). The HF centerline is represented by the top horizontal boundary, while the inlet and outlet of the lumen are represented by the left and right boundaries, respectively. According to the simulation results, in the absence of bHb/PolyHb in the circulating cell culture media, the majority of space within the HF is hypoxic, with the pO2 level generally below 20 mm Hg. Oxygenation of the HF bioreactor gradually improves with an increase in PolyHb concentration. Our data shows

80 that L-PolyHbs with different G:Hb molar ratios exhibited similar O2 transport capabilities compared to bHb and Oxyglobin®, which substantially reduced the volume of the hypoxic space in the HF. H-PolyHbs supported a much lower potential to oxygenate the HF even at a heme concentration of 100%.

Figure 3.8: ECS Zonation with [Heme] = 100%. 100% [Heme] = 15 g/dL of bHb. The biophysical parameters used to simulate 50:1 L- and 40:1 H-PolyHb were taken from the literature [132].

These results are consistent with the ECS zonation plot, which is designed to show further details of the distribution of O2 within the cell-containing ECS (Figure 3.8).

Oxygenation within the ECS was quantified into the following pO2 zones [240]: hyperoxic (> 70 mm Hg), periportal (60-70 mm Hg), pericentral (35-60 mm Hg), perivenous (20-35 mm Hg) and hypoxic (< 20 mm Hg) zones. As mentioned previously, the computed hypoxic region (pO2 < 20 mm Hg) dominates ~95% of the ECS without supplementation of bHb/PolyHbs and only a small percentage of hepatocytes (~5%) are subjected to in vivo pO2 levels (ranging between 20-70 mm Hg). It is clearly apparent that

81 L-PolyHbs greatly enhanced O2 transport inside the ECS based on the simulation results.

In the presence of L-PolyHbs, the entire ECS volume is in the pO2 range 20-70 mm Hg and the volume of the hypoxic region diminishes significantly, which is similar to the effect of bHb and Oxyglobin®. In contrast, a large percentage of the volume in the ECS remained hypoxic with supplementation of H-PolyHbs in the circulating cell culture media. In fact, the volume of the ECS exposed to hypoxic pO2 levels (< 20 mm Hg) increased with increasing G:Hb molar ratio for H-PolyHbs.

3.4 Discussion:

This chapter is aimed at investigating the effect of bHb O2 saturation and G:Hb molar ratio on the biophysical properties and oxygenation potential of PolyHb solutions.

In this work, bHb was polymerized in either the fully deoxygenated (0% fractional O2 saturation, Y=0) or fully oxygenated (100% fractional O2 saturation, Y=1) states with

G:Hb molar ratios ranging from 10:1 to 30:1.

Glutaraldehyde is known to non-specifically react with several reactive moieties on amino acid residues of proteins [241] including primary amine groups on lysine [242], phenolic rings on tyrosine [243], imidazole rings on histidine [244] and sulfhydryl groups on cysteine [244] to generate cross-linked proteins with heterogenous MWs [133]. The degree of intermolecular cross-linking is a function of the glutaraldehyde to protein molar ratio [245].

In our study, the MW of PolyHbs increased with increasing G:Hb ratios from 10:1 to

30:1 (Table 3.1). At a low G:Hb molar ratio (10:1), the SDS- and native-PAGE showed that the PolyHbs fractions were primarily intramolecularly cross-linked with a smaller

82 portion possessing intermolecular cross-links (Figures 3.2 and 3.3). On the other hand, at higher G:Hb molar ratios (20:1 and 30:1), more intramolecular cross-linking sites reacted with free glutaraldehyde, which may have sterically hindered further intramolecular cross-linking, but facilitated intermolecular cross-linking/polymerization. This is also supported by the SDS- and native-PAGE of the 20:1 and 30:1 PolyHbs, which showed strong bands above 250 kDa and diffuse smearing from 50 to 250 kDa (Figures 2 and 3).

The MW of an HBOC is an important parameter to consider when evaluating its efficacy and safety for a particular clinical application [70, 95, 126, 190, 246-248]. In this study, the hydrodynamic diameter of PolyHbs increased with increasing MW as expected.

Although the molecular diameter of unmodified bHb is ≥ 7 nm [238], which satisfies the baseline size requirement of 7 nm for an HBOC in order to prevent its extravasation through blood vessel walls [126]. However, unmodified bHb cannot be used as an HBOC since it will easily dissociate into harmful  dimers in the blood stream [92], thereby facilitating its extravasation through blood vessel walls and promotion of oxidative tissue stress [249], scavenging of NO [108, 250], overoxygenation of surrounding tissues [95] as well as development of vasoconstriction and systemic hypertension. The PolyHbs synthesized at the G:Hb molar ratio of 20:1 and 30:1 possessed larger hydrodynamic diameters than 20 nm, which can circumvent the risks associated with tissue extravasation.

Sakai et al. have shown that the degree of vasoconstriction and hypertension elicited by a wide range of HBOCs was inversely proportional to the MW (size) of the HBOC

[174]. In our lab, we validated these same heuristics for a series of L-PolyHbs with varying MW [190]. In a previously published study, we observed that ultrahigh MW 50:1

83 L-PolyHb displayed no vasoconstriction and elicited the lowest increase in mean arterial pressure compared to other variable MW L-PolyHbs [190]. In contrast, we observed that

PolyHbs with MWs < 500 kDa elicited substantial vasoconstriction and hypertension compared to PolyHbs with MWs > 500 kDa [190]. In light of these results, PolyHbs with

MWs greater than 500 kDa should be used for transfusion applications. Therefore, it is predicted that in vivo administration of 10:1 L- and H-PolyHbs will lead to harmful vasoconstrictive and hypertensive effects although their hydrodynamic diameter were larger than 7 nm. In contrast, the weight average molar mass (MW) of 20:1 L- and H-

PolyHb is 749 and 1025 kDa, respectively, and should not elicit substantial vasoconstriction and hypertension upon transfusion. Thus, a G:Hb molar ratio of at least

20:1 should be used in the future in order to produce PolyHbs with high enough MW for use in transfusion medicine.

The ζ potential of a colloidal particle is an indicator of the surface electrostatic repulsive force of the particle. Therefore, the ζ potential of a colloidal particle could predict its possibility to aggregate with neighboring colloidal particles in solution [251].

The greater the absolute value of the ζ potential, the more stable is the PolyHb solution.

When administrated in vivo, the ζ potential of PolyHb will be affected by the pH and various ions in blood. Thus, in this research, PBS (pH 7.4) was used to evaluate the ζ potential of PolyHbs, since it has a similar ion concentration to that of plasma. An ideal

O2 carrier should have a ζ potential which is similar to or higher in absolute value than that of RBCs in order to reduce/eliminate undesirable aggregation. The pI of bHb is 6.8

[252]. Therefore, in PBS (pH 7.4), bHb and PolyHbs possessed negative surface charges.

However, the ζ potential of PolyHbs didn’t increase proportionally with increasing MW.

84 For example, the average MW of 30:1 L-PolyHb is approximately 1330 kDa which is as big as 20 Hb tetramers. But the ζ potential of it was only slightly higher than twice that of bHb. This can be explained by two reasons. First, steric hindrance resulting from the cross-links between bHb with glutaraldehyde can mask the surface charges on PolyHb.

Second, the reaction of glutaraldehyde with bHb can modify the side chains of surface amino acid residues possessing negative charges. The ζ potential of all PolyHbs in PBS were within the limited flocculation range of |5| to |15| mV, suggesting a low potential for aggregation [251] in vivo. Interestingly, L-PolyHbs possessed higher ζ potentials (> |10| mV) than H-PolyHbs. Thus, L-PolyHb dispersions should be more colloidally stable than

H-PolyHb dispersions in solution. Among all of the PolyHbs in this study, the ζ potential of 30:1 L-PolyHb was -14.7 mV, which is close to that of RBCs (-16.8 mV [253]) suggesting that the 30:1 L-PolyHb solution should be the most colloidally stable PolyHb solutions in vivo.

The O2 affinity and cooperativity coefficient of L-PolyHb solutions are low compared to bHb, suggesting that polymerization of bHb in the T-state constrains the resulting L-PolyHbs more in a deoxy-conformation compared to unmodified bHb. The cooperativity of L-PolyHb decreases with increasing G:Hb molar ratio. This is because the intra- and inter-molecular cross-links reduce the structural flexibility of PolyHb in a concentration dependent manner. Hence, the quaternary conformational changes which otherwise would occur during normal O2 binding are restricted by the presence of the chemical cross-links. This leads to a loss of cooperative binding of O2 for all PolyHbs compared to unmodified bHb. In this study, 10:1 L-PolyHb yielded a P50 value of approximately 29 mm Hg, which was close to that of unmodified bHb (27 mm Hg). This

85 can be explained by the existence of unreacted Hb tetramer in the PolyHb solution since these molecules cannot pass through the 100 kDa TFF cartridge [211, 254]. In contrast, the P50 of 20:1 L- (37 mm Hg) and 30:1 L-PolyHb (41.1 mm Hg) are similar to that of

HBOC 201®, which is 38 mm Hg [141, 255], suggesting that these two types of L-

® PolyHbs may have similar O2 transport capabilities to HBOC 201 . Interestingly, the P50 of 30:1 L-PolyHb is also similar to that of 50:1 L-PolyHb (41 mm Hg) synthesized in a previous publication [132], indicating that the P50 of L-PolyHbs does not increase with

G:Hb molar ratio in an unrestricted manner. The P50s of L-Polyhbs are lower than that of

O-raffinose polymerized human Hb Hemolink® (Hemosol Inc, Toronto, Canada), which is 47~52 mm Hg [127, 183, 256]. This is because the oxidized O-raffinose cross-linking reagent completely freezes Hb in the T-state [183].

In contrast to L-PolyHbs, all H-PolyHb solutions displayed very high O2 affinities, indicating that they are constrained in a more oxy-conformation than unmodified bHb.

The 10:1 H-PolyHb solution yielded a P50 of 4.5 mm Hg. Consequently, it was observed that the O2 affinity increased with increasing G:Hb molar ratio. This is consistent with reports that indicate that the O2 affinity increases with increasing degree of polymerization in an oxygenated environment [43, 175, 176, 257, 258]. In addition, H-

PolyHb has a lower P50 than that of the amide bond polymerized (―zero-link‖) bHb

Oxyvita® (6.4 mm Hg [129, 259]), indicating that modification with glutaraldehyde cross- linking has more influence on the O2 affinity than amide bond mediated cross-linking.

Interestingly, the cooperativity coefficients of all H-PolyHbs with G:Hb molar ratios ranging from 10:1 to 30:1 display a similar value of approximately 0.6 irrespective of the

G:Hb molar ratio. This phenomenon has also been observed in Hemolink® (n = 0.6-1)

86 [260, 261]. This is probably due to the higher reactivity of glutaraldehyde with R-state

Hb versus T-state Hb [176]. Thus, the steric hindrance caused by intra- and intermolecular cross-links that hampers transmission of any quaternary changes in the H-

PolyHb superstructure should be stronger as to reduce the cooperativity to a lower level than that of L-PolyHb or unmodified bHb. Previously, Guillochon et al. [175] observed that the cooperativity of glutaraldehyde modified Hb dropped to 1.0 at a G:Hb molar ratio of 4.8:1. Therefore, it is expected that H-PolyHbs synthesized at higher G:Hb molar ratios should possess lower cooperativity coefficients.

All the L- and H-PolyHb solutions in this study exhibited metHb levels lower than

6%, which satisfy the 10% metHb criterion necessary for a Hb-based transfusion solution

[88, 131]. Interestingly, the metHb level of all PolyHb solutions in our study was not sensitive to the G:Hb molar ratio. This is ascribed to the preventative measures that were taken in our polymerization methodology. First, NaCNBH3 was used to reduce the metHb level after the polymerization reaction [132]. Second, PolyHb solutions were diafiltrated against modified lactated Ringer’s buffer containing N-acetyl-L-cysteine, which is an antioxidant able to retard the oxidation of heme [228]. The overall metHb levels of H-

PolyHb solutions are a little higher than that of L-PolyHb solutions. This makes sense since the H-PolyHbs were prepared in an O2 saturated environment, which made the heme groups of bHb more vulnerable to autoxidation during polymerization.

At a Hb concentration (10 g/dL), close to physiological levels (14 g/dL), the viscosity of both L- and H-PolyHbs increased with increasing G:Hb molar ratio and displayed higher viscosities than unmodified bHb (1.6 cp) or blood (~ 3 cp) [193]. The

87 increase in viscosity with increasing G:Hb molar ratio is consistent with the increased

MW of the PolyHb macromolecule.

High viscosity HBOCs may be beneficial in blood transfusion for 2 reasons. First, transfusion of high viscosity solutions can improve the functional capillary density [198], which primarily determines in vivo survival after transfusion [197]. In addition, transfusion of high viscosity solutions increases blood vessel wall shear stress, which induces the endothelium to produce the vasodilator NO [191, 199, 200, 202, 203, 262] which will thereby alleviate vasoconstriction and hypertension elicited by NO scavenging of HBOCs. Second, high viscosity solutions will retard the diffusion of PolyHb macromolecules in the blood to reduce both the concentration of PolyHb near the vessel wall and facilitated diffusion of excess O2 to the vessel wall.

The COP is another vital property that a HBOC must possess in order to assess its suitability for transfusion and determines the fluid balance between the intra- and extra- vascular space [43]. The COP of both L- and H-PolyHbs in our study decreased with increasing G:Hb molar ratio. This is because the COP depends on the number of colloidal particles in solution [43]. Therefore at the same total protein concentration in solution, the number of PolyHb particles present in solution is inversely proportionally to the

PolyHb MW. Therefore, the COP of both 10:1 L- and H-PolyHbs and 20:1 L-PolyHbs

(Table 3.3) are higher than that of normal blood (~ 27 mm Hg), while the COP of

PolyHbs with higher G:Hb molar ratios substantially dropped to 2 mm Hg for 30:1 L- and 14 mm Hg for 30:1 H-PolyHb. Among all the PolyHbs in our study, the 20:1 L-

PolyHb solution (24 mm Hg) showed a COP which is closest to that of normal blood.

Thus, 20:1 L-PolyHb should enable simultaneous O2 transport and blood volume

88 expansion with a slight adjustment of the COP by human serum albumin (HSA) or hydroxyethyl starch (HES) [202]. Although transfusion solutions with higher COPs were considered to cause the in-flow of fluid from the tissue space into the intravascular compartment [43], Wettstein et al. [198] reported that transfusion of hyperoncotic and hyperviscous HES in hemorrhagic shock facilitated rapid recovery of the microcirculation by increasing blood volume in a short time frame while maintaining cardiac output. In light of this published report, 20:1 H-PolyHb with a MW of 1025 kDa,

P50 of 2.15 mm Hg and COP of 39 mm Hg may be useful in oxygenating the hypoxic tissues of patients suffering from hemorrhagic shock.

Fast kinetic analysis of gaseous ligand binding showed that L-PolyHb solutions

possessed higher koff, O2 values than unmodified bHb, while H-PolyHb solutions possessed

lower koff, O2 values, which were generally reflected by their high O2 affinities. The koff, O2 values for L-PolyHbs increases and eventually plateaus off with increasing G:Hb molar

-1 -1 ratio. Both 20:1 and 30:1 L-PolyHbs have similar koff, O2 values of 58.6 s and 57.1 s , respectively, which is higher than that of 10:1 L-PolyHbs (47.2 s-1), comparable to that of

Oxyglobin® (61.8 s-1) [263] but smaller than that of O-raffinose polymerized human Hb

-1 (O-R-PolyHbA0) (130 s ) [127]. The CO association rate constant is another indicator of the O2 affinity of PolyHb solutions. For L-PolyHb solutions, their kon,CO values are slightly lower than that of bHb and decreases with increasing cross-link density, which is in agreement with their declining O2 affinity and reflected by their P50s. In contrast, H-

PolyHbs showed a dramatic elevation in kon, CO by at least 20-fold, which is consistent with the large drop in P50, confirming that polymerization of Hb with glutaraldehyde plays a significant role in regulating the O2 affinity of PolyHbs. The calculated O2 89 association rate constants of L-PolyHbs decreased with decreasing O2 affinity, which is

in the same trend observed for kon, CO, while kon, O2 for H-PolyHbs increased with increasing O2 affinity. However, the O2 association rate constants of L-PolyHbs increased with increasing pO2 while those of H-PolyHbs decreased with increasing pO2, which can be attributed to the negative cooperativity of H-PolyHbs. In addition, although a

remarkable difference exists among the P50s of unmodified bHb and PolyHbs, the kon, O2 values of all PolyHbs are comparable and much lower than that of unmodified bHb, indicating that O2 binding to PolyHb is not only a function of O2 affinity, but is also affected by other factors such as the cooperativity coefficient. Compared with the kon, CO

value for bHb, the kon, O2 value for unmodified bHb is much higher, indicating that O2 binds to bHb faster than CO does, which is consistent with previous studies in the

literature [264, 265]. Similarly, the kon, O2 values of L-PolyHbs are also higher than their kon, CO values. However, the kon, CO values of H-PolyHbs show no change as a function of

O2 affinity or G:Hb molar ratio and are higher than their kon, O2 values, demonstrating that kon, CO is mainly determined by the PolyHb O2 affinity and eventually plateaus off at high

O2 affinity. The NO dioxygenation rate constants remain almost unchanged among all

PolyHbs and unmodified bHbs, which is consistent with a previous report that suggest that the extent of NO scavenging in vivo was almost the same for different molecular configurations of HBOC including PolyHb, PEG-Hb and cross-linked Hb [70].

In the absence of intracellular reducing enzymes, ferrous Hb (Fe2+) can rapidly

3+ autoxidize into metHb (Fe ) which is cytotoxic [84-87] and unable to transport O2 [72].

Thus, the autoxidation of Hb is considered a source of metHb [131] and an important factor affecting the efficacy and safety of HBOCs upon transfusion into the systemic 90 circulation. In our study, the autoxidation rate constants of PolyHb are higher than that of unmodified Hb (Table 3.5), which is consistent with the results of previous research on cross-linked bHb [129, 131, 183, 263]. Interestingly, the redox potentials of two commercial cross-linked Hbs (Hemolink® and Oxyglobin®) were found to be higher than that of unmodified bHb [266], indicating that the polymerized Hbs should be less able to be oxidized than bHb. On the other hand, it has been reported that the autoxidation of heme was directly related to the extent of exposure of the heme to its neighboring aqueous environment [267]. Thus, the observed higher autoxidation rate constants of

PolyHb solutions could be attributed to the alteration of the quaternary conformation of the PolyHb superstructure caused by intra- and intermolecular cross-links of bHb with glutaraldehyde which rendered the heme groups in the PolyHb superstructure more accessible to the aqueous bulk environment compared to unmodified bHb [266]. H-

PolyHbs possess lower autoxidation rate constants compared to L-PolyHbs, which may be due to the fact that polymerization of bHb in the R-state caused more steric hindrance to gaseous ligands compared to polymerization in the T-state since bHb exhibited higher reactivity with glutaraldehyde in the R-state [176]. Rapid autoxidation of PolyHb solutions can be of great concern when evaluating the clinical application of PolyHb in transfusion medicine. Thus, further research should be conducted with regards to this aspect of PolyHb biochemistry in order to retard the undesirable autoxidation reaction.

In a HF bioreactor, the continuously circulating medium can deliver nutrients and O2 to cultured cells, while simultaneously carrying away metabolite waste products, which to a certain extent mimics the function of blood vessels [232]. Therefore, a simple O2 transport model was developed to preliminarily investigate the ability of the PolyHbs

91 synthesized in this study to oxygenate cultured cells/tissues. Compared to unmodified bHb, it was computationally calculated that all PolyHbs exhibited slightly lower O2 consumption rates at lower inlet pO2s (< 40 mm Hg) (Figure 3.6). This is probably due to the low cooperativity and diffusivity of these macromolecules. However, there is not much difference between all PolyHb solutions and unmodified bHb in terms of the simulated normalized O2 consumption rate at higher inlet pO2s (~150 mm Hg). In general, the hypoxic region in the ECS shrinks as the P50 of the PolyHb increases in magnitude.

The results of the computer simulations showed that oxygenation of the HF bioreactor is a function of many factors such as HBOC concentration, HBOC O2 affinity, HBOC diffusivity, inlet pO2, flow rate and the geometry of the reactor. After comparing the oxygenation results between all HBOCs with the simulation parameters fixed at the same values, we predicted that L-PolyHbs (high P50) enhanced O2 transport into the ECS of the

HF bioreactor in a similar manner compared to the commercial HBOC Oxyglobin®, irrespective of the G:Hb molar ratio. However, under the simulated conditions studied

(inlet pO2 = 80 mm Hg), the size of the pericentral zone (35-60 mm Hg) decreased with increasing G:Hb molar ratio, while the size of the perivenous zone (20-35 mm Hg) increased with increasing G:Hb molar ratio, suggesting that the oxygenation capabilities of L-PolyHbs does not strictly increase with increasing P50. This may be because the ultrahigh MW of PolyHb molecules limit their diffusion in solution, which in turn reduces the amount of O2 released to the HF ECS. Therefore, this suggests that in order to obtain optimum oxygenation of tissues not only is the P50 an important factor, but also molecular size must be taken into account to get a balance between these two properties.

However, for H-PolyHbs which have very high O2 affinities, the O2 consumption rate is

92 calculated to be higher than that of unmodified bHb and L-PolyHbs only under extremely hypoxic conditions in which the pO2 level is close to zero, suggesting that H-PolyHbs cannot offload their store of O2 until extensive hypoxia occurs. Hence, H-PolyHbs may be useful in oxygenating hypoxic tissues under extreme duress.

Therefore for both transfusion and tissue engineering applications, L-PolyHbs will oxygenate surrounding tissues better than H-PolyHbs. One can envision a transfusion scenario where it would be beneficial to transfuse a mixture of L- and H-PolyHbs into a patient suffering from hypoxia, in which the H-PolyHb would first initiate oxygenation of hypoxic tissues which would then be followed up by oxygenation of normoxic tissues with L-PolyHb. Hence in light of these O2 transport simulations, it is recommended that either pure L-PolyHb or mixtures of L- and H-PolyHbs be used for transfusion and tissue engineering applications. However, the results of this study do not support the administration of pure H-PolyHb for either application.

3.5 Conclusions:

The effect of varying the G:Hb molar ratio on the biophysical properties of PolyHbs synthesized in either the fully deoxygenated or fully oxygenated state was systematically investigated in this study. The MW of the resulting PolyHbs increased with increasing

G:Hb molar ratio. Increasing the G:Hb molar ratio also reduced the O2 affinity and CO association rate constants of L-PolyHbs, while it led to higher O2 affinity and significantly increased the CO association rate constants for H-PolyHbs compared to bHb.

The metHb level and NO dioxygenation rate constants were observed to be insensitive to the G:Hb molar ratio for all PolyHbs. In addition, all PolyHbs exhibited higher viscosities

93 but lower COPs compared to bHb, which also varied as a function of the G:Hb molar ratio. Low O2 affinity L-PolyHbs were predicted to be able to better oxygenate a HF bioreactor compared to high O2 affinity H-PolyHbs. Taken together, these findings may be helpful in the future design of PolyHbs for use in tissue engineering and transfusion medicine.

94 CHAPTER 4

Simulation of NO and O2 Transport Facilitated by PolyHb Solutions in an Arteriole

4.1 Introduction:

Nitric oxide (NO) is an important messenger molecule in vivo, which plays an important role in many physiological processes such as regulation of cellular respiration

[268, 269] and vascular tone [270], angiogenesis [271, 272], wound healing [273], leukocyte-mediated inflammation [274] and nervous system signaling [275]. NO is mainly generated by the layer of endothelial cells that comprise the blood vessel wall

[276-278]. When NO diffuses into the smooth muscle cell layer of the blood vessel wall,

NO can activate soluble guanylate cyclase (sGC) and induce the relaxation (i.e. dilation) of the blood vessel [276]. In the blood, NO can react rapidly with heme-containing macromolecules such as Hb encapsulated inside RBCs, acellular cell-free Hb and HBOCs

[108] forming nitrate and metHb. Thus, RBCs, cell-free Hb and HBOCs are usually considered as NO sinks [279]. To further complicate matters,  dimers and tetrameric

Hb itself can extravasate out of the lumen of blood vessels through pores in the blood vessel wall, where these molecules can scavenge NO thereby eliciting vasoconstriction and systemic hypertension [279], which is one of the main side-effects hampering the clinical use of the currently commercially available HBOCs [142-145, 185].

94 Generation of NO from the endothelial layer can be affected by various factors such as the shear stress acting on the blood vessel wall and oxidative stress [94]. Both experimental studies on in vitro cell cultures and animal models as well as mathematical simulations have observed and predicted that increasing the wall shear stress on the endothelium can increase the production rate of NO [199, 280-284]. In the physiological range of observed shear stresses ranging from 6-25 dyne/cm2, the rate of release of NO was found to be linearly dependent on the shear stress acting on the blood vessel wall

[285, 286]. Compared to the commercial HBOC Oxyglobin® (1.8 cP at 13 g/dL) [287], the viscosity of L- and H-PolyHbs synthesized in our study are much higher than that of

Oxyglobin® (3.6 - 14 cP at 10 g/dL). Transfusion of 50:1 L- and 40:1 H-PolyHb solutions elicited little to mild hypertension and vasoconstriction, despite possessing NO dioxygenation rate constants that were similar to that of cell-free Hb. Thus, it is reasonable to hypothesize that our L- and H-PolyHb solutions should be able to enhance the shear stress-induced production of NO, due to their high solution viscosity, which should offset the rapid NO scavenging capability of acellular HBOCs.

Therefore in this chapter, a mathematical model of simultaneous NO and O2 transport in the presence of PolyHb can help us to better understand and evaluate the potential efficacy and safety of PolyHb upon transfusion and optimize future design of

PolyHbs. In a previous study by our group, a NO/O2 transport model was developed to investigate NO/O2 transport in an arteriole and its surrounding tissues facilitated by various types of HBOCs [211]. However, shear stress-induced generation of NO was not taken into account, which in turn underestimated NO production upon transfusion of

HBOC. In addition, to our knowledge, a mathematical model of simultaneous NO/O2

95 transport in vivo facilitated by ultrahigh MW PolyHbs has never been conducted to date.

We hypothesize that our PolyHb formulations should help to facilitate an increase in tissue oxygenation upon transfusion, while maintaining the NO concentration in the blood vessel wall and smooth muscle layer.

Therefore in this chapter, we propose to develop an improved transport model to investigate the oxygenation potential of PolyHb and evaluate the effect of shear stress caused by highly viscous PolyHb solutions on NO transport. The commercially available polymerized HBOC product Oxyglobin® will be simulated for comparison.

4.2 Computational Methods

A mathematical model based on modification of the Krogh tissue cylinder (KTC) model [211, 236] was used to simulate mass transport of NO and O2 in a single arteriole to surrounding tissue upon transfusion of PolyHbs (Figure 4.1 A). The geometry of the arteriole and surrounding tissue will be divided into 7 regions: 1) the RBC-rich core

(0≤r≤r1); 2) the RBC-poor plasma layer (r1

(G) (r2

(IS) (r4

(TS) (r6

60 μm. In this model, RBCs and PolyHb flow through the arteriolar region (0≤r≤r2) and enter the arteriole at Z=0 and exit it at Z= Lc. O2 is transported by the convective flow of dissolved O2, oxygenated RBCs and PolyHbs as well as other HBOCs in the blood. Due to the O2 gradient originating from the blood to the tissues, O2 diffuses through the blood vessel wall into the endothelial cell layer, smooth muscle layer and tissue space where it

96 is consumed by these 3 cell types, while NO is generated only from the endothelial cell layer and diffuses into both the blood and surrounding tissues. Extravasation of PolyHb molecules through the blood vessel wall and vasoconstriction following transfusion of the

HBOCs were not considered in this model. This mathematical model was numerically simulated using the Chemical Engineering module of COMSOL Multiphysics (Comsol,

Burlington, MA).

Figure 4.1. Model Geometry of Arteriole. A: Side-view of the 500-μm-long arteriole and surrounding tissue used in the simulation. B: Schematic cross-section of the modified KTC model geometry delineating each subregion: 1) RBC-rich core (0≤r≤r1); 2) RBC-poor plasma layer (r1

4.2.1 Hb-O2 Release/Binding Kinetics

The kinetics of gaseous ligand binding and release between dissolved O2 and Hb inside RBCs/HBOCs is reversible and can be derived from a simplified reaction between one O2 molecule and one heme group (equation 4.1). Thus, all calculations in this chapter will be based on the amount of heme groups present in RBCs and HBOCs (equation 4.2).

97 The total amount of Hb is the sum of oxyHb (HbO2) and cell-free deoxygenated Hb

(equation 4.3), and the saturation of Hb with O2 is defined as S (equation 4.4). The rate of

formation of O2 from HbO2 is described by equation 4.5, where koff,O2 and kon,O2 are the O2

dissociation and binding rate constants of Hb, respectively. At equilibrium when RO2 is

zero, kon,O2 is given by equation 4.6, where Ye is the saturation of Hb with O2 at equilibrium (4.7), where a1~a4 are the adair constants for either RBCs and HBOCs. After

reorganizing the equations, the expression for RO2 can be rewritten in the form of equation

4.8. Therefore, the corresponding rates of formation of O2 from dissociation of O2 from

HbO2 inside RBCs and HBOCs are given as equations 4.9 and 4.10, respectively.

The rate of formation of HbO2 is given by RHbO2 = -RO2. The rate of formation of

HbO2 in RBCs and HBOCs are described by equations 4.11 and 4.12, respectively.

kon, O2  O22 Hb HbO (4.1) koff, O2

[HBOC ]overallMWHBOC 4 [ HBOC ] overall [Heme ]HBOC    4  (4.2) MWHBOC MW bHb MW bHb

[Hb ]total =[ Hb ] [ HbO2 ] (4.3)

[]HbO S  2 (4.4) []Hb total

RO,,2 =koff O 2 [ HbO22 ]  k on O 2  [ O ]  [ Hb ] (4.5)

kYoff, O2  e kon, O2  (4.6) [OY2 ] (1-e )

a pO 2 a  pO2  3 a  pO 3  4 a  pO 4 Y  1 2 2 2 3 2 4 2 (4.7) e 2 3 4 41 a1  pO 2  a 2  pO 2  a 3  pO 2  a 4  pO 2 

98 kYoff, O 2  e RO,22 =koff O  [ Hb ]  S   [ Hb ]  (1  S ) total(1Y ) total e (4.8) Ye koff, O 2 [ Hb ]total  [ S   (1  S )] 1Ye

Ye, RBC RO2,RBC k off, O 2  RBC [ HbRBC ]total  [ S RBC   (1  S RBC )] (4.9) 1Ye, RBC

Ye, HBOC RO2,HBOC k off, O 2  HBOC [ HBOC ]total  [ S HBOC   (1  S HBOC )] (4.10) 1Ye, HBOC

Ye, RBC RHbO22 -RBCkoff , O RBC  [Hb RBC ]total  [  (1  S RBC )  S RBC ] (4.11) 1Ye, RBC

Ye, HBOC RHbO22 -HBOCkoff , O HBOC  [ HBOC ]total  [  (1  S HBOC )  S HBOC ] (4.12) 1Ye, HBOC

4.2.2 Mass Balance on O2/NO with Hb Encapsulated within RBCs and HBOCs

4.2.2.1 Arteriole Lumen (0≤r≤r2)

In the arteriole lumen, blood can be roughly considered as a solution of RBCs and the RBC concentration can be described as a function of the hematocrit (hct), which is the percentage of blood volume occupied by packed RBCs [288]. However, due to the

Fåhraeus effect, RBCs tend to migrate towards the central axis of the blood vessel, which generates a RBC-rich core with higher Hb concentration and a RBC-poor plasma region with lower Hb concentration [289, 290]. In the RBC-rich core, the local hct is higher than the discharge hct (HD) and assumed to be a constant value (HC), while in the RBC-poor plasma region, the hct decreases from the core region toward the blood vessel wall and can be described by different concentration profiles ranging from step, linear and parabolic profiles [284, 291]. Chen et al. [291] investigated the effect of these 3 hct 99 profiles on NO/O2 transport by mathematical modeling and observed that the simulation results obtained by using a linear profile to describe the hct in the RBC-poor region was in good agreement with experimental data. Thus, in this chapter, a linear hct profile and blood convection rate vz will be used to define the local flow velocity and hct at a given radius (r) (equations 4.13 and 4.14 where Uavg is the average flow velocity of blood). HC will be calculated by performing a general mass balance on RBCs in the arteriole lumen

(equation 4.15). Thus, the local concentration of Hb from RBCs ([HbRBC]total) is given

by[][]HbRBC total Hb RBC overall hct, while the luminal HBOC concentration ([HBOC]total) at a given radius will be determined by equation 4.16.

 Hc  0 rr1 hct() r   rr2  (4.13) Hc r12 r r  rr21

 Uavg  0 rr1 vz   rr2  (4.14) Uavg r12 r r  rr21

rr22 2hct ( r ) vz rdr 2 H D v z rdr (4.15) 00

1 hct [][]HBOCtotal HBOC overall (4.16) 1 HD

O2 in the arteriole lumen will be transported by convection and diffusion in the plasma and will reversibly bind to heme groups in RBCs and HBOCs. Thus, the mass balance for the formation of O2 inside the lumen is described by equation 4.17, where α is

z the solubility of O2 in aqueous media, DO2 is the diffusivity of O2. z '() is the Lc

r dimensionless length and r '() is the dimensionless radius where Rc is the radius of Rc

100 pO2 arteriole. []'()pO2  is the dimensionless local O2 partial pressure, where P50-RBC is P50RBC

vz the P50 of RBCs and vz '() is the dimensionless blood flow velocity. Similarly, Uavg mass balances of luminal RBCs and HBOCs are given by equations 4.18 and 4.19, in which DHb and DHBOC represent the diffusivity of Hb inside RBCs and diffusivity of

HBOC molecules, respectively. On the other hand, NO is transported via convection and diffusion but scavenged by Hb in a first-order reaction (i.e. NO dioxygenation reaction)

(equation 4.20). Therefore, the mass balance of NO is shown in equations 4.21 and 4.22,

[]NO where []'NO  , DNO is the diffusivity of NO while the kox,NO is the NO []NO0 dioxygenation reaction. The oxidation reaction between NO and O2 is neglected due to the low reaction rate in comparison to the reaction rate of the NO dioxygenation reaction

[292].

 Uavg  P50RBC  v z ' O2 ' 2 Ye, RBC  DO2 P 50['[]'] pO2  koff , O2  RBC  [ HbRBC ][ total  S RBC  (1 S RBC )] Lc z'1 Ye, RBC (4.17)

Ye koff, O2  HBOC [ HBOC ]total  [ S RBC   (1  S RBC )] 1Ye

USavg RBC 2 Ye, RBC vz',   D Hb RBC [  SRBC ]  k off O2 RBC  [  (1  S RBC )  S RBC ] (4.18) Lc  z1 Ye, RBC

USavg HBOC 2 Ye, HBOC vz',   D HBOC [  SHBOC ]  k off O2  HBOC  [  (1  S HBOC )  S HBOC ] (4.19) Lc z1 Y e, HBOC

kox NO Hb NO  HbNO (4.20)

0 Uavg [][]' NO NO 02 vz'  [ NO ]  D NO  [  '[]NO ']  R NO (4.21) Lzc 

RNO k ox, NO RBC []'[][]'[] NO  Hb RBC total  k ox, NO HBOC  NO  HBOC total (4.22)

101 4.2.2.2 Glycocalyx Layer (r2

The glycocalyx layer is considered a macromolecular mesh lining the surface of the luminal side of the endothelial cell layer [293], which is structurally composed of a complex network of polysaccharides and associated proteins such as albumin [294]. The thickness of the glycocalyx layer is estimated to be 0.5 μm [295]. As mentioned earlier, extravasation of Hb/HBOC through the blood vessel wall will be neglected in this model.

Thus, O2 and NO simply diffuse through this region without reacting with Hb inside

RBCs or PolyHb. The transport of O2 and NO are described by equations 4.23 and 4.24.

2 0 P50 RBC  D O2  [  ' [ pO 2 ]'] (4.23)

02 0 [NO ]  DNO  [  '[]'NO ] (4.24)

4.2.2.3 Endothelial Cell Layer (r3

In the endothelial cell layer, the NO production rate was represented by RNO’ while the O2 consumption rate was assumed to be twice the NO production rate [296, 297]

(equation 4.25). NO is generated proportional to the shear stress on the blood vessel wall in the range of (6~25 dyne/cm2) [286] (equations 4.26~27), where Q is the volumetric flow rate (ml/s) and η is the apparent viscosity. The basal release rate of NO is assumed to be 5.3*10-12mol*cm-2s-1 at a normal blood vessel wall shear stress of 24 dyne/cm2

(Shear_con) under the action of a physiologically normal blood velocity of 0.5 cm/s and apparent blood viscosity of 3.0 cp in an arteriole with diameter of 50 μm [298], while the shear stress upon transfusion of HBOCs is represented by Shear_Exp.

RRO2 2*NO ' (4.25)

102 Shear_ Exp RRNO'' NO, control (4.26) Shear_ con

4* *Q Shear _ exp  (4.27)  * R3

The endothelial layer in this chapter is assumed to have a thickness of 1 μm and length of 500 μm. Thus, the control NO release rate (RNO, control’) is estimated to be 106 nM/s.

4.2.2.4 Interstitial Layer (r4

Similar to the glycocalyx layer, there is no Hb or RBCs present in the interstitial layer. O2 and NO simply diffuse through this region and their transport is described by equations 4.28 and 4.29.

2 0 P50 RBC  D O2  [  ' [ pO 2 ]'] (4.28)

02 0 [NO ]  DNO  [  '[]NO '] (4.29)

4.2.2.5 Smooth Muscle Cell Layer (r5

O2 and NO diffuse into smooth muscle cell layer and are consumed by the smooth muscle cells. The consumption of O2 by smooth muscle cells is controlled by M-M kinetics and inhibited by the presence of NO (equation 4.30), where Qmax,sm is the maximum O2 consumption rate constant and Ksm is the M-M kinetic constant without NO.

The consumption of NO by the smooth muscle cells follows first order kinetics (equation

4.31) with kNO-sm as the first order NO reaction rate constant.

2 []'pO2 0 P50 RBC  D O2  [  ' [ pO 2 ]']  Q max, sm  (4.30) []'NO [pO2 ]' ( Ksm (1 )) 0.027M 103 02 0 [NO ]  DNO  [  '[]'NO ]  k NO sm  [ NO ]' (4.31)

4.2.2.6 Tissue Space (r6

In the tissue space, O2 and NO are transported by diffusion and are consumed by the tissue cells. Similarly, the consumption of O2 in the tissue space follows M-M kinetics and is inhibited by the presence of NO (equation 4.32), where Qmax,ts is the maximum O2 consumption rate constant in the tissue space and Kts is the M-M kinetic constant in the tissue space in the absence of NO. The consumption of NO in the tissue space follows first-order kinetics (equation 4.33, where kNO-ts is the first order NO reaction rate constant.

2 []'pO2 0 P50 RBC  D O2  [  ' [ pO 2 ]']  Q max, ts  (4.32) []'NO [pO2 ]' ( Kts  (1  )) 0.027M

02 0 [NO ]  DNO  [  '[]'NO ]  k NO ts  [ NO ]' (4.33)

4.2.3 Model Parameters

Table 4.1 Constants and Parameters used in the Simulations.

Simulation Symbol Value Units Ref Parameter Lc Length of arteriole 500 μm [211] Rc Radius of arteriole 30 μm [287] Uavg Average blood flow rate 0.42 cm/s [287] [HbRBC]total_g Concentration of Hb in RBC 34 g/dL [211] Molar Concentration of Hb in [Hb ] 21250 μM [211] RBC overall RBC † HD Discharge hct 11% [287] and 18% [287]

0.13 for HD 11% Calculated from equation HC Hct of RBC-rich core 0.20 for HD 18% 4.15 [NO]0 Basal NO concentration 0.01 μM [211] αO2 (blood) Solubility of O2 in blood vessel 1.34 µmol/(L*mm Hg) [291] αO 2 Solubility of O in glycocalyx 1.34 µmol/(L*mm Hg) [291] (glycocalyx) 2 Solubility of O in endothelial αO (endo) 2 1.34 µmol/(L*mm Hg) [291] 2 cell layer Solubility of O in interstitial αO (interstitial) 2 1.10 µmol/(L*mm Hg) [299] 2 layer Solubility of O in smooth αO (smooth) 2 1.34 µmol/(L*mm Hg) [291] 2 muscle cell layer

104 Table 4.1 Continued

αO2 (tissue) Solubility of O2 in tissue 1.52 µmol/(L*mm Hg) [291]

17.7 (HD 11%) r1 Radius of RBC-rich core µm Extrapolated from ref [300] 19.8 (HD 18%) Outer radius of RBC-poor r 29.5 µm [291] 2 region r3 Outer radius of glycocalyx 30 µm [291] r4 Outer radius of endothelial layer 31 µm [291] r5 Outer radius of interstitial layer 31.5 µm [291] Outer radius of smooth muscle r 37.5 µm [291] 6 layer r7 Outer radius of tissue 135 µm [291] -5 -5 2 DO2 Diffusivity of O2 2.8*10 or 1.4*10 cm /s [291], [301] -5 -5 2 DNO Diffusivity of NO 3.3*10 or1.65*10 cm /s [291], [301] Overall concentration of PolyHb [HBOC] 3.5 or 10 g/dL [287] overall in vivo Maximum O consumption rate Q 2 1 µM/s [291] max,sm constant in smooth muscle M-M constant of O K 2 1 mm Hg [291] sm consumption in smooth muscle First order NO reaction rate k 0.05 1/(µM*s) [94, 302] NO-sm constant in smooth muscle Maximum O consumption rate Q 2 20 µM/s [291] max,ts constant in tissue M-M constant of O K 2 1 mm Hg [291] ts consumption in tissue First order NO reaction rate k 0.05 1/(µM*s) [94, 302] NO-ts constant in tissue †: personal communication from Prof. Pedro Cabrales, Department of Bioengineering, University of California, San Diego, CA.

The constants and parameters used in this model are listed in Table 4.1. The arteriole diameter, average blood flow rate and discharge hct (HD) of this simulation were taken from reference [287] and personal communication from Dr Cabrales: the HD was set to 11% for animal studies conducted using 50:1 L- and 40:1 H-PolyHb solutions and the HD was set to 18% for animal studies conducted using 20:1 and 30:1 L- and H-

PolyHb solutions. The radius of the RBC-rich region (r1) changes proportionally with the

HD [300, 303] and the r1 used in this chapter is extrapolated from values of r1 measured at hcts of 20%, 30% and 40% that are present in the literature [300]. For the calculation of

NO transport, an inlet pO2 of 60 mm Hg was chosen to mimic the inlet pO2s reported in the literature [287]. For the simulation of O2 transport facilitated by HBOCs, inlet pO2 values were varied from 10-150 mm Hg to simulate both hypoxic and normal 105 physiological conditions. The diffusivities of O2 (DO2) and NO (DNO) in the solid phase

(i.e. endothelial layer, smooth muscle cell layer and tissue space) were reported to be half of the values reported in free solution [304, 305]. Thus in this chapter, DO2 and DNO in these regions were assumed to be half of the values reported in blood phase. The overall concentration of HBOCs used in these simulations was estimated from the literature

[287].

Table 4.2 Physical Properties of PolyHbs and other HBOCs.

P50 -1 -1 -1 Solutions n k off, O (s ) k (M s ) Ref (mm Hg) 2 ox, NO [299], RBCs 29.3 2.2 4.4 [306] 0.14 [302] 20:1 L-PolyHb 37.1 1.05 58.6 17.1 [307] 20:1 H-PolyHb 2.18 0.6 29.7 16.4 [307] 30:1 L-PolyHb 41 1.01 57.1 18.7 [307] 30:1 H-PolyHb 1.84 0.69 24.7 17.5 [307] 40:1 H-PolyHb 0.66 0.57 22 17.5 [307] 50:1 L-PolyHb 41 0.87 53 18.9 [132] Oxyglobin® 38 1.2 60 15 [178] bHb 27 2.7 36.1 18 [307, 308]

Table 4.3 Diffusivity and Adair Constants of PolyHbs and other HBOCs.

Adair parameters Solutions Diffusivity (cm2/s) Ref a1 a2 a3 a4 RBCs f(Hb) (equ 4.34) 2.59*10-3 1.77*10-3 1.86*10-11 1.39*10-6 [211] a 20:1 L-PolyHb 2.01*10-7 1.03*10-1 4.21*10-3 7.52*10-5 5.27*10-7 a 20:1 H-PolyHb 1.74*10-7 14.139 7.7835 2.753 4.17*10-2 a 30:1 L-PolyHb 1.54*10-7 9.73*10-2 3.43*10-3 5.41*10-5 3.1*10-7 a b 30:1 H-PolyHb 7.57*10-8 8.2511 7.5548 6.4383 5.98*10-1 a 40:1 H-PolyHb 3.91*10-8 9.0158 24.717 27.613 1.1931 a 50:1 L-PolyHb 4.84*10-8 1.47*10-1 5.20*10-3 8.48*10-5 3.40*10-7 a Oxyglobin® 3.69*10-7 3.39*10-2 2.49*10-3 2.79*10-5 6.55*10-7 [178] a bHb 6.21*10-7 6.977*10-3 2.153*10-3 5.000*10-6 1.884*10-6 [178] a: HBOC diffusivity was calculated using equation 3-8. b : Adair parameters were curve fitted from O2-HBOC equilibrium curves given in references [132, 307].

106 The physical properties of PolyHbs and RBCs used in this chapter are listed in Table

4.2 and 4.3. Diffusivities of PolyHbs and other HBOCs were calculated using equation

3.8, while the diffusivity of Hb encapsulated within RBCs was estimated using equation

4.34 [299].

RBC total [R]_*Hb BC total g hct 7 []_*Hb g hct 128 DHb 9.74*10 *(1 )*10 (4.34) 46

Table 4.4 Blood Viscosity and Blood Vessel Wall Shear Stress After Transfusion.

Blood Shear stress Solution Viscosity (cP) (dyne/cm2) 11% hct 1.5 8.4 18% hct 1.9 10.9 20:1 L-PolyHb 4.1† 23.3 20:1 H-PolyHb 3.8† 21.6 30:1 L-PolyHb 4.6† 26.2 30:1 H-PolyHb 4.4† 25.1 40:1 H-PolyHb 2.5[287] 15.2[287] 50:1 L-PolyHb 2.9[287] 16.8[287] 1.5 (11% hct) 8.4 (11% hct) Oxyglobin® 1.9 (18% hct) 10.9 (18% hct) 1.5 (11% hct) 8.4 (11% hct) bHb 1.9 (18% hct) 10.9 (18% hct) †: experimental results were obtained from Prof. Pedro Cabrales, Department of Bioengineering, University of California, San Diego.

The viscosities of blood after transfusion with PolyHbs and other HBOCs were taken from reference [287] and experimental results. The blood vessel wall shear stress upon transfusion of 20:1 and 30:1 PolyHbs were calculated using equation 4.27 and listed in

Table 4.4, while those of 40:1 H and 50:1 L PolyHb were taken from the experimental results of reference [287]. The viscosity of blood at hcts of 11% and 18% were estimated from the literature [309]. The effect of transfusion of Oxyglobin® and bHb on the viscosity of blood was neglected in this study, since Oxyglobin® [287] and bHb [307]

107 possess viscosities of 1.8 and 1.6 cp, respectively, even at concentrations higher than 10 g/dL.

4.3 Results:

4.3.1 Effect of PolyHb on NO Profiles

Vasoconstriction caused by the transfusion of commercial HBOCs due to the undesired rapid scavenging of NO by HBOCs has long been one of the major side-effects hampering the clinical use of HBOCs in transfusion medicine [142-144, 250, 310, 311].

However, it has been found that transfusion of ultrahigh MW PolyHb solutions with high viscosity exhibits less vasoactivity upon transfusion compared to the commercial PolyHb

Oxyglobin® [190, 287]. This is partly because administration of high viscosity solutions increases blood vessel wall shear stress, which induces the endothelium to produce the vasodilator NO [191, 199, 200, 202, 203, 262] so as to offset the intrinsic NO scavenging effect of HBOCs. Thus, in this chapter, we are also going to study the NO generating effect of transfusion of high viscosity PolyHb solutions by computer simulation. In order to compare our results with experiments in the literature [287], the total PolyHb concentration in our calculations will be set to 3.5 g/dL and the inlet pO2 will be set to be

60 mm Hg.

The effect of increasing the blood vessel wall shear stress due to the transfusion of high viscosity PolyHb solutions on the steady-state NO distribution at hcts of 11% and

18% are shown in Figure 4.2 and compared to the NO profiles without considering the influence of blood vessel wall shear stress. Hemodiluted blood with a hct of 11% [287] and 18% in the absence of HBOCs were used as controls. The NO profiles displayed in

108 Figure 4.2 represent the NO concentration distribution at the axial midpoint of the arteriole (z = 250 μm) and at an inlet pO2 = 60 mm Hg. The results indicate that although the simulated steady-state NO concentration profile upon transfusion of PolyHb solutions were lower than that of the control at both hcts considering the effect of shear stress or not, the NO-inducing effect of blood vessel wall shear stress greatly increased the overall steady-state concentration of NO across the region spanning the arteriole to surrounding tissue space compared to the case of not considering the NO-inducing ability of blood vessel wall shear stress. In addition, the overall computed steady-state NO concentration profile upon transfusion of ultrahigh MW PolyHbs was much higher than that of either

Oxyglobin® or cell-free bHb.

Figure 4.2: The effect of increasing blood vessel wall shear stress upon transfusion of PolyHb solutions on the NO concentration profiles at pO2 inlet = 60 mm Hg: A): NO profile at hct = 11%; B): NO profile at hct = 18%. The vertical dashed lines represent the RBC-rich region, RBC-poor region, glycocalyx, endothelial cell layer, interstitial layer, smooth muscle cell layer and tissue space, respectively from left to right.

109

Figure 4.3: The average NO concentration in the smooth muscle layer. A): Hct = 11%, L 50:1’ and H 40:1’ represent the groups that did not consider shear stress-induced production of NO by the endothelium; B): Hct = 18%, L 20:1’, L 30:1’ and H 20:1’, H 30:1’ represent the groups that did not consider shear stress- induced production of NO by the endothelium.

The average NO concentration in the smooth muscle layer of the control and

PolyHb-transfusion groups was calculated and compared in Figure 4.3. Similarly, the control groups had the highest average NO concentration at the two hcts with the NO

110 concentration at a hct of 18% slightly higher than that at a hct of 11% due to the higher wall shear stress at a hct of 18%. However, when considering the NO-inducing effect of blood viscosity, the average NO concentration of the PolyHb-transfusion groups reached

50% (hct = 11%) and 60-70% (hct = 18%) of the control group regardless of the P50 and n of the PolyHb, while the NO concentration of no-shear-stress PolyHb solutions,

Oxyglobin® and cell-free bHb groups were only about 20-30% of the control groups, indicating that the NO-inducing effect due to the induction of higher blood vessel shear stress caused by the transfusion of high viscosity PolyHb solutions could offset the harmful NO scavenging effect of HBOCs. Figure 4.4 shows the relationship between the blood vessel wall shear stress and the average NO concentration in the arteriole region

(Figure 4.4.A) and the endothelial layer (blood vessel wall region) (Figure 4.4.B) for all the HBOCs groups. In both regions, the average NO concentration increased with blood vessel wall shear stress in an almost linear fashion regardless of the kox, NO values.

Figure 4.4: The relationship between blood vessel wall shear stress and average NO concentration in the arteriole (A) and endothelial layer (B) of all PolyHb groups.

111 4.3.2 pO2 Profiles Upon Transfusion of Hb/PolyHb solutions

Figure 4.5: Steady state pO2 profiles upon transfusion of PolyHb at hct = 11% and varying inlet pO2. A-D): PolyHb = 3.5 g/dL, inlet pO2 = 10, 60, 100, 150 mm Hg; E-H): PolyHb = 10 g/dL, inlet pO2 = 10, 60, 100, 150 mm Hg. The vertical dashed lines represent the RBC-rich region, RBC-poor region, glycocalyx, endothelial cell layer, interstitial layer, smooth muscle cell layer and tissue space, respectively from left to right.

112

Figure 4.6: Steady state pO2 profiles upon transfusion of PolyHb at hct = 18% and varying inlet pO2. A-D): PolyHb = 3.5 g/dL, inlet pO2 = 10, 60, 100, 150 mm Hg; E-H): PolyHb = 10 g/dL, inlet pO2 = 10, 60, 100, 150 mm Hg. The vertical dashed lines represent the RBC-rich region, RBC-poor region, glycocalyx, endothelial cell layer, interstitial layer, smooth muscle cell layer and tissue space, respectively from left to right.

Figures 4.5 and 4.6 display the steady-state O2 tension profile under varying inlet pO2s at the midpoint of the arteriole (z = 250 μm) after transfusion of PolyHb solutions, 113 with Oxyglobin® and cell-free bHb as negative controls. Diluted blood with a hct of 11%

[287] and 18% in the absence of HBOC was used as a control. The inlet pO2 levels were chosen to represent hypoxia (10 mm Hg), in vivo experimental conditions during hemodilution (60 mm Hg), normal physiological pO2 in an arteriole before hemodilution

(100 mm Hg) and supraphysiological levels of O2 (150 mm Hg). The HBOC concentration used in the simulation was set to 3.5 g/dL [287] which was the total HBOC concentration in 35% exchange transfusion and 10 g/dL which is close to normal physiological Hb concentration [312]. NO production was assumed to be identical for both PolyHb concentrations. First, at both hcts, transfusion of L-PolyHb solutions increased the in vivo oxygen tension from the arteriole to the tissue space at all inlet pO2 levels in a dose dependent manner especially in the RBC-poor region of the arteriole. In addition, transfusion of HBOCs increased the pO2 profiles higher at a hct of 11% than at a hct of 18%, suggesting that L-PolyHb solutions could improve in vivo O2 transport under conditions of serious blood loss. However, too high of a PolyHb concentration may cause excessive rise in blood viscosity, which can cause a viscosity dependent rise in blood flow resistance and systemic hypertension [282]. Thus, more experiments should be conducted to optimize the transfusion concentration of L-PolyHb to be used in a clinical setting. Second, simulated pO2 profiles are affected by the O2 affinity of the

PolyHb solution, which affects the O2 dissociation rate. All H-PolyHb solutions (P50s ranging from 0.66~2 mm Hg) could improve O2 transport to the same level as L-PolyHb

® solutions (P50s ranging from 37~41 mm Hg), Oxyglobin (P50 of 38 mm Hg) and cell- free bHb (P50 of 27 mm Hg) under hypoxic conditions (inlet pO2 = 10 mm Hg) regardless of PolyHb size and hct. However, H-PolyHbs exhibited less ability to improve O2

114 transport than either L-PolyHb and Oxyglobin® at experimental hemodilution (60 mm

Hg), normal physiological or supraphysiological (100 and 150 mm Hg) inlet pO2 levels

(Figure 4.5 B/C/D/F/G/H and Figure 4.6 B/C/D/F/G/H). In fact, the higher the O2 affinity of the H-PolyHb solution, the lower its ability to improve O2 transport upon transfusion. These results are in agreement with the prediction in chapters 2 and 3, which showed that H-PolyHb solutions with extremely high O2 affinity and low O2 dissociation rate constant can potentially oxygenate hypoxic tissues, which is similar to that of MP4

[107, 150], but is not effective under normoxic oxygenation conditions. In contrast, all L-

PolyHb solutions possessed an enhanced ability to improve O2 transport in the tissue space compared to H-PolyHb solutions and cell-free bHb and could increase the O2

® tension to a similar level as Oxyglobin at all PolyHb concentrations and inlet pO2s.

These computer simulation results confirm the prediction that L-PolyHbs have the same

® O2 transport ability as Oxyglobin . In addition, cell-free bHb exhibited less ability to

® improve O2 transport than either L-PolyHb and Oxyglobin at high inlet pO2s (100-150 mm Hg, Figure 4.5.D/G/H, Figure 4.6.C/D/G/H) and was able to improve O2 transport no better than 40:1 and 30:1 H-PolyHbs, which had the highest O2 affinities among the

HBOCs studied, at an inlet pO2 of 150 mmHg at a hct 18% especially at high concentrations (Figure 4.5.H and Figure 4.6.H). This phenomenon can be ascribed to

-1 two reasons. First, bHb had lower O2 dissociation rate constant (36.1 s ) compared to that of PolyHbs (53-58 s-1) and Oxyglobin® (60 s-1). Second, because of the high cooperativity of bHb (2.7), bHb has higher O2 affinity at high inlet pO2 compared to the

L-PolyHb and Oxyglobin®.

115 4.3.3 O2 Transfer Rate of PolyHbs

The O2 transfer rates at steady-state after administration of HBOC solutions at varying inlet pO2s from 10-150 mmHg were calculated as the difference in the O2 flux between the entrance and exit of the arteriole lumen and shown in Figure 4.7 in

® comparison with those of Oxyglobin and cell-free bHb. The O2 transfer rates of RBCs in diluted blood at hcts of 11% and 18% were used as controls. At both hcts, the O2 transfer rates of all simulation groups increased with increasing inlet pO2 and reached a plateau when the inlet pO2 was higher than 130 mm Hg. At a hct of 11% and at all inlet pO2s

(Figure 4.7 A), L- and H-PolyHbs exhibited higher calculated O2 transfer rates than that of the control and any other HBOC. Interestingly, although H-PolyHb didn’t enhance oxygenation of the tissue as effectively as L-PolyHb, 40:1 H-PolyHb had a slightly higher O2 transfer rate than 50:1 L-PolyHb did at inlet pO2s lower than 60 mm Hg, while

L-PolyHb transferred O2 faster at inlet pO2s higher than 70 mm Hg. In contrast,

® Oxyglobin and bHb had lower O2 transfer rates than the control at inlet pO2s lower than

70 mm Hg. When the inlet pO2 was higher than 70 mm Hg, the O2 transfer rates of

Oxyglobin® and bHb rose to a similar level to that of H-PolyHb, which was consistent with the lower oxygenation ability of H-PolyHb. Similarly, at a hct of 18% (Figure

4.7.B), two H-PolyHbs had similar O2 transfer rates, which were higher than that of the control and any of the other HBOCs at low to medium inlet pO2s (10-70 mm Hg). The O2 transfer rates of both L-PolyHbs rose to a higher level than the control when the inlet pO2 was higher than 60 mm Hg and to a similar level to H-PolyHb at an inlet pO2 higher than

® 70 mm Hg. Again, the O2 transfer rates of Oxyglobin and bHb were the lowest when the inlet pO2 was lower than 80 mm Hg and rose to a higher level compared to the control at

116 an inlet pO2 higher than 80 mm Hg. Interestingly, when the inlet pO2 was lower than 70-

80 mm Hg, the O2 transfer rates were inversely related to the HBOC concentration at both hcts, while at an inlet pO2 between 80-120 mm Hg, O2 transfer rates increased with

HBOC concentration at a hct of 11% but mainly remained unaffected by concentration at a hct of 18%.

117

Figure 4.7: O2 transfer rate across the arteriole at different hcts and inlet pO2s. A): O2 transfer rate at a hct of 11% at [HBOC] = 3.5 g/dL and 10 g/dL, respectively. B): O2 transfer rate at a hct of 18% at [HBOC] = 3.5 g/dL and 10 g/dL, respectively.

118 4.4 Discussion:

This chapter is aimed at investigating the effect of PolyHb’s biophysical properties such as O2 affinity and viscosity on the transport of NO and O2 to primarily evaluate the efficacy and safety of PolyHbs in clinical applications via a mathematical model based on modification of the KTC model. In order to replicate the structure of an arteriole and blood flow, the physiological parameters of an arteriole such as hct, average blood flow rate, diameter of arteriole lumen and blood viscosity after transfusion were all taken from animal test results on PolyHb solutions [287]. The effect of endothelial derived NO production induced by the high wall shear stress generated by the flow of high viscosity

PolyHb solutions on NO transport was also investigated in this study.

In vivo, NO is mainly generated in the endothelial cell layer through enzymatic synthesis by nitric oxide synthase (NOS) [276] and subsequently diffuses into the smooth muscle cell layer activating guanylate cyclase to regulate vascular dilation [109]. On the other hand, NO can be rapidly scavenged by heme-containing macromolecules such as

Hb and HBOCs [108] forming nitrate and metHb when it enters the blood circulation.

Hence, the concentration profile of NO across the arteriole lumen to surrounding tissues should be determined by the balance of the NO scavenging rate by heme-containing macromolecules and the NO production rate of the endothelial cell layer.

When injected into the blood, high viscosity PolyHb solutions tend to elevate blood and plasma viscosity, which was previously considered to be harmful to the organism, since blood viscosity were reported to be related to the physiopathology of hypertension and heart disease [283, 313-316]. However, recent experiments and a mathematical model have demonstrated that slight elevation of blood viscosity, e.g. a slight increase in

119 the hct by less than 19%, could lower the MAP due to the increased generation of blood vessel wall shear stress-mediated production of the vasodilator NO [203, 282, 284]. The

PolyHb solutions studied in this chapter increased the apparent blood viscosity after transfusion but didn’t overly surpass the normal physiological blood viscosity (Table 4.3).

Thus, transfusion of high viscosity PolyHb solutions is expected to increase the production of NO without excessively increasing blood flow resistance. On the other hand, all PolyHb solutions have high NO dioxygenation rate constants compared to

RBCs. Therefore, the NO concentration profile in an arteriole will be affected by the combined effects of the aforementioned two factors.

Equation 4.27 was used to estimate the blood vessel wall shear stress after transfusion using in vivo apparent blood viscosity, blood flow rate and arteriole vessel size data that was presented in the literature [287].

Table 4.5 Comparison of Estimated Blood Vessel Wall Shear Stress Against Measured Blood Vessel Wall Shear Stress.

Calculated Shear Stress Measured Shear Blood sample 2 (dyne/cm ) Stress (dyne/cm2) Baseline (hct 48%)[287] 26.2 27.5 Normal (hct 45%) [298] 23.9 24 50:1 L-PolyHb[287] 16.5 16.8 40:1 H-PolyHb[287] 14.2 15.2

Table 4.4 lists the blood vessel wall shear stress reported in the literature with the calculated results using equation 4.27. It is evident that the calculated results are in good

120 agreement with the experimental results. Thus, equation 4.27 was accurate in estimating the blood vessel wall shear stress for this simulation.

In our simulations (Figures 4.2 and 4.3), the two controls without HBOC had the highest NO concentration profiles across the arteriole into the tissue space at each respective hct. Interestingly, the [NO] profiles and average NO concentrations of the two control groups in the arteriole lumen and smooth muscle layer (Figures 4.2 and 4.3) increased with increasing hct as expected, although there were more RBCs present in the lumen of blood vessels at a hct of 18% to consume NO, indicating that when the NO consumption rate is low, enhancement in the NO production rate can offset the increased

NO consumption by the increased hct. For PolyHbs, although their computationally calculated NO profiles were lower than those of the controls no matter whether the blood vessel wall shear stress was taken into account or not, the NO concentration profile upon transfusion of PolyHb solutions was observed to be much higher than that of Oxyglobin® and cell-free bHb. Transfusion of PolyHb solutions increased the NO release rate from the blood vessel wall 2-2.5 times compared to that of the control, since the NO production rate is linearly dependent on the blood vessel wall shear stress [285] within the physiological wall shear stress range of 6-25 dyne/cm2 [286]. However, the NO dioxygenation rate constant (kox,NO) of PolyHbs and other HBOCs are two orders of magnitude higher than that of RBCs. This explains why the NO concentration profiles of the controls were higher than that of the PolyHb solutions and other HBOCs which displayed much lower NO production rates. However, this result demonstrated that the enhanced NO production rate due to transfusion of high viscosity PolyHbs offset the NO scavenging effect of the PolyHbs to a large extent. The calculated NO concentration

121 profile upon transfusion of PolyHb solutions in the smooth muscle layer was proportionally to the blood vessel wall shear stress and could reach about 70% of the control values when the NO-inducing effect of high wall shear stress due to the high viscosity of PolyHb solutions was considered, while that of Oxyglobin® was about 30% of the control (≤ 30 nM). Condorelli et al. reported that the median activation concentration of sGC by NO is around 23 nM [317]. Upon transfusion of PolyHb solutions, the simulated NO concentration inside the smooth muscle layer ranged from 40

- 60 nM. Thus, transfusion of PolyHb solutions is expected leave enough NO in the smooth muscle layer for vasodilation compared to Oxyglobin®. This is consistent with published results of animal tests with PolyHb solutions, in which a little to mild rise in

MAP was observed after transfusion of the >500 kDa fraction of ultrahigh MW PolyHb solutions [190, 287], while transfusion of Oxyglobin® and PolyHb fractions smaller than

500 kDa with lower viscosity (1.0~1.4 cp) elicited a significant rise in MAP [143, 190,

287]. Additionally, it is expected that the extremely low diffusivities of PolyHbs in solution due to their ultrahigh MW could retard the scavenging of NO by PolyHb in the arteriole lumen. However, simulation results showed that the NO concentration in the arteriole lumen was lower than 0.2 nM due to the high NO dioxygenation rate, which masked the effects of PolyHb diffusivity. In summary, according to our simulation results, the [NO] profile is primarily a function of the blood vessel wall shear stress-mediated NO production rate and the rate of NO scavenging, with the NO-inducing effect of blood vessel wall shear stress playing a more important role than the NO consumption rate. The shear stress increases proportionally with the viscosity of blood, which depends on the concentration of PolyHb in the blood. However, an excessive increase in the viscosity of

122 blood, e.g. higher than 50%, may offset the vasodilatory effect of shear stress-induced

NO production via the viscosity dependent rise of blood flow resistance, which may cause hypertension [282]. Thus, further efforts must be devoted to optimize the PolyHb concentration in order to not only maintain the vasodilatory effect of shear stress-induced

NO production but also avoid high viscosity dependent hypertension. In addition, more research should be conducted to lower the reactivity of PolyHb with NO, while maintaining a slightly high solution viscosity.

The pO2 profiles are reported to be affected by the hct profile, velocity profile, radial distribution of HBOC and P50 [291]. Simulation results of the radial pO2 distribution

(Figures 4.5 and 4.6) showed that transfusion of HBOC increased the pO2 profiles most efficiently in the RBC-poor region of the arteriole at all inlet pO2s and hcts. This is because most HBOC molecules were present in the RBC-poor region (equation 4.16) due to the behavior of the hct profile. Hereby, more O2 was released in this area than in the

RBC-rich core. However, although the concentration of HBOC in the RBC-poor region increased with increasing r according to equation 4.16, the pO2 dropped rapidly from the

RBC-rich core to the glycocalyx layer instead of increasing with HBOC concentration due to the fast diffusion of O2 into the neighboring endothelial cell layer where O2 was rapidly consumed by the cells.

Comparing the simulation results using different HBOCs at varying concentrations and inlet pO2s with in vivo measurements, all HBOCs only slightly improved oxygenation of the surrounding tissues, while either 50:1 L-PolyHb or 40:1 H-PolyHb could increase the average pO2 in the arteriole by 30-60 mm Hg [287]. This discrepancy between simulation and experiments may be because in the model the HBOC are

123 assumed to be equilibrated with the inlet pO2, while in the in vivo studies the PolyHb solutions, which had been equilibrated with room air (pO2 ≈ 160 mm Hg), may contain large amounts of O2 upon transfusion. Thus, the HBOC in the simulation can be much less O2 saturated than those in the in vivo experiments, especially the L-PolyHbs, and the inlet concentration of O2 carried by blood in the simulation can be much lower than that of the in vivo experiments so that the amount of O2 which can be offloaded to tissues may be underestimated in the simulation.

The O2 affinity which is quantified by the P50 is another important factor that determines the efficacy of PolyHb solutions. Under hypoxic conditions (e.g. 10 mm Hg,

Figure 4.5.A/E and Figure 4.6.A/E), H-PolyHb could obviously improve oxygenation of the surrounding tissues as efficiently as other HBOCs, while under normoxic conditions

H-PolyHb could hardly increase the pO2 profile. The efficiency of tissue oxygenation displayed by high O2 affinity PolyHb solutions in hypoxic regions is in agreement with results of previous animal tests and simulations with low P50 HBOCs in hypoxic tissues such as capillaries. However, there is debate on the efficacy of low P50 HBOCs to oxygenate tissues under normoxic conditions. Sakai et al. [318] and Cabrales et al. [319] reported that transfusion of vesicle encapsulated Hb with high O2 affinities oxygenated tissues better than vesicles with low O2 affinities at an inlet pO2 of 60 mm Hg. Tsai et al.

[205] reported that MP4 (P50 ≈ 5 mm Hg) could release more O2 in the capillaries than

Oxyglobin®. On the other hand, recently, Cabrales et al. [287] observed in animal tests that high O2 affinity H-PolyHb was much less effective in oxygenating tissues surrounding arterioles than L-PolyHb. Our simulation results support the in vivo results of PolyHb. The discrepancy between our simulation results and in vivo results of

124 transfusion of MP4 and high O2 affinity Hb vesicles may be caused by the different intrinsic biophysical properties of these HBOCs.

In contrast, all L-PolyHbs could obviously increase the in vivo pO2 profiles to a

® similar level to that of Oxyglobin at all inlet pO2s (Figures 4.5 and 4.6) irrespective of the cross-link density. The tissue oxygenation abilities of 20:1 and 30:1 PolyHbs were almost identical, which is consistent to their similar O2 affinities and O2 dissociation rates.

However, the pO2 profiles of free bHb were slightly higher than those of L-PolyHbs at inlet pO2s of 0 and 60 mm Hg at both hcts (Figures 4.5 A/B/E/F and 4.6), while the pO2

® profiles of Oxyglobin were also higher than that of L-PolyHb at inlet pO2s ranging from

10 to 100 mm Hg at a hct of 11% (Figure 4.5.A/B/C/G/E/F, Figure 4.6A/B/C/G/E/F).

This phenomenon is similar to O2 transport simulation results obtained in hepatic hollow fiber bioreactors [307]. These results indicated that at low to medium in vivo pO2s, the diffusivity of HBOC molecules can have more of an influence on blood vessel and tissue oxygenation than the O2 affinity and O2 dissociation rate constant indicating that the limited diffusion of PolyHb due to its ultrahigh MW may slightly retard offloading of O2 to tissues. Therefore, these simulation results suggest that although high MW PolyHbs can help to eliminate the harmful extravasation of HBOC through the blood vessel wall and prolong the in vivo circulation time, the MW should engineered to obtain optimum tissue oxygenation and the longest circulation time at all blood vessel inlet pO2 levels.

Compared with the controls, Oxyglobin® and free bHb, both L- and H-PolyHbs displayed higher O2 transfer rates (Figure 4.7). Patton and Palmer [299] predicted in a model of O2 transport in a capillary that the release of O2 from RBCs and HBOCs is affected by the HBOC/RBC O2 affinity, cooperativity and HBOCs can compete with

125 RBCs for O2 and behave as sink for O2 depending on the O2 saturation of the RBCs and

HBOCs. Our simulation results showed that the O2 consumption rate, which was affected by the NO production rate, played a more important role in regulating the rate of O2

® delivery. For example, bHb has a higher O2 saturation than L-PolyHb and Oxyglobin at any pO2, but lower O2 transfer rate than those HBOCs under low to medium inlet pO2s.

® In our simulations, Oxyglobin and bHb had a lower O2 transfer rate than the control and

PolyHbs at inlet pO2s ranging from 20 - 60 mm Hg. In this low inlet pO2 range, HBOCs were partially O2 saturated, leaving unoccupied O2 binding sites in the arteriole lumen.

Patton and Palmer [299] suggested that when the tissue including endothelial layer, smooth muscle cell and surrounding tissue had low O2 consumption, unconsumed O2 could diffuse back into the arteriole lumen and bind to any available O2 binding sites there. In our simulations, the NO production rate determines the O2 consumption rate in

® the endothelium (equation 4.25). Thus, Oxyglobin and bHb possessed lower O2 consumption rates compared to PolyHbs and behaved more like an O2 sink. Free bHb

® exhibited this O2 sink effect more so than Oxyglobin in this inlet pO2 range, since the higher cooperativity of bHb (2.7) made bHb more readily able to bind O2. The O2 ―sink‖ effect of these HBOCs can be more pronounced at higher HBOC concentrations when more HBOC is available to bind O2. Therefore, the diffusion of O2 in an arteriole especially in the RBC-poor plasma region can be retarded by HBOCs in a dose dependent manner at lower inlet pO2 levels. When the inlet pO2 was higher than 70 mm Hg, the O2

―sink‖ effect of the HBOCs started to be gradually counteracted by the increasing amount of transported O2 and the high O2 dissociation rate constants of these HBOCs. Therefore, the HBOCs O2 transfer rates increased significantly and surpassed that of the control,

126 when the inlet pO2 was higher than 70 mm Hg. On the other hand, O2 diffusion from the plasma layer to the blood vessel wall could be accelerated by 2-2.5 fold after transfusion of L- and H-PolyHbs compared to transfusion of other HBOCs due to the high NO production rate in the endothelial layer, which also increased the release of O2 from

PolyHbs regardless of their O2 affinity and saturation. Therefore, PolyHbs displayed

® higher O2 transfer rates compared to the control and the commercial HBOC Oxyglobin or bHb.

4.5 Conclusions:

NO and O2 transport in the presence of RBCs and HBOCs under anemic conditions was studied in this chapter with a mathematical model. According to the simulation results, administration of high viscosity ultrahigh MW PolyHb solutions can have less vasoconstriction by promoting blood vessel wall shear stress dependent generation of the vasodilator NO especially in the vessel walls compared to the commercial PolyHb

Oxyglobin®. However, quenching of NO by PolyHb molecules in the arteriole lumen is inevitable due to the high NO dioxygenation rate constant of the PolyHb solution. Under hypoxic conditions, all PolyHbs could improve tissue oxygenation under anemic conditions as effectively as the commercial HBOC Oxyglobin®, while L-PolyHb solutions were more effective in delivering O2 than H-PolyHb solutions under normoxic conditions. In addition, all ultrahigh MW PolyHb displayed higher O2 transfer rates than the commercial HBOC Oxyglobin®. Taken together, the simulation results indicate that a safe and efficacious HBOC should possess high viscosity and a low NO dioxygenation rate constant.

127 CHAPTER 5

Conclusions and Future Work

5.1 Summary and conclusions

The primary objective of this dissertation was to synthesize and characterize a small library of variable MW glutaraldehyde PolyHbs with two distinct O2 affinities as safe and efficacious O2 carriers for use in transfusion medicine. We hypothesized that increasing the size of the PolyHb molecule can decrease both the extravasation of PolyHb through the pores in the blood vessel wall and scavenging of NO by the endothelium, which can help to alleviate the post-transfusion vasoconstriction in the microcirculation, systemic hypertension as well as oxidative damage to tissues and organs. Ultrahigh MW PolyHbs possess reduced diffusion coefficients compared to small MW commercial PolyHbs and cell-free Hb, which will reduce the rate of PolyHb transport to the surface of the blood vessel wall and therefore dually reduce the rates of NO scavenging and HBOC-facilitated overtransport of O2.

In this dissertation, we examined our hypothesis both experimentally and theoretically. By controlling the O2 tension at either 0 or over 749 mmHg during the polymerization reaction and employing high molar ratios of glutaraldehyde to bHb, we synthesized PolyHb solutions possessing ultrahigh MWs and distinctly low (L-PolyHb) or high O2 (H-PolyHb) affinities. The remaining bHb tetramers and small oligomers of bHb were cleared from the final product via tangential flow filtration (TFF). The MW of

128 all PolyHbs increased with increasing cross-link density, while H-PolyHb solutions possessed higher MWs than L-PolyHb solutions polymerized at the same cross-link density. The O2 affinity of L-PolyHb solutions increased with increasing cross-link density while that of H-PolyHb solutions was inversely related to increasing cross-link density. Stopped-flow measurements showed that L-PolyHb solutions possessed much higher O2 releasing rate constants compared to unmodified cell-free bHb and RBCs. In addition, all PolyHbs possessed low metHb levels and transfusion of these PolyHbs indicated limited PolyHb dissociation in vivo, indicating that the ultrahigh MW PolyHb prepared in this research represents a suitable basis for future HBOC development.

In order to preliminarily investigate the ability of PolyHbs to oxygenate tissues and evaluate the influence of PolyHb on the transport of NO, mathematical models were developed to simulate the O2 transport in a hollow fiber hepatic bioreactor and combined

NO/O2 transport in an arteriole based on the Krogh tissue cylinder model. In both the bioreactor and tissue cylinder model, L-PolyHb solutions were observed to deliver O2 more efficiently than H-PolyHb solutions, while H-PolyHb solutions can only offload O2 under hypoxic conditions, indicating that H-PolyHb solutions should be used together with L-PolyHb for transfusion or tissue engineering applications. Simulation of NO and

O2 transport in an arteriole showed that although scavenging of endothelial-derived NO by PolyHb solutions was unavoidable, the increased blood vessel wall shear stress due to transfusion of the high viscosity of PolyHb solutions maintained the blood vessel wall

NO concentration to a similar level to that of the control without HBOC transfusion and much higher than that of the commercial HBOC Oxyglobin®, indicating that PolyHb solutions can serve as safe transfusion solutions that minimally disturb NO homeostasis.

129

5.2 Future work

Although the ultrahigh MW PolyHb solutions synthesized in this work have the potential to be used as an O2 carrier in transfusion medicine, more research needs to be conducted before it can be widely administered in clinical trials. From the experimental results shown in this work, the autoxidation rate constants of PolyHb solutions are higher than that of RBCs and unmodified cell-free bHb. Hence, further work should be done to engineer PolyHb solutions to be more stable in oxidative environments. In addition, more effort should also be devoted to the scale-up of PolyHb solutions in order to facilitate their clinical use.

5.2.1 Conjugation of PolyHb with antioxidant enzymes

Due to the open conformation of the heme pocket towards the bulk aqueous environment caused by the glutaraldehyde cross-links, the autoxidation rate constants of all PolyHb solutions were higher than that of unmodified bHb [266], which can be a source of harmful metHb and reactive oxygen species (ROS) when transfused in vivo.

Thus, reducing the autoxidation rate constant of PolyHb solutions can help to enhance the safety of PolyHb solutions. It has been demonstrated that supplementing Hb solutions with reducing enzymes such as superoxide dismutase (SOD) and catalase (CAT) can scavenge ROS and H2O2 and reduce the rate of autoxidation [131]. Based on this concept, bHb were co-polymerized or cross-linked with SOD and CAT yielding PolyHb-SOD-

CAT conjugates [138, 320, 321], which were effective in reducing ROS in vivo after administration. Considering that direct co-polymerization of bHb with other proteins may

130 affect the final MW and other biophysical properties of the PolyHb solution, we propose to conjugate PolyHb with these enzymes in order to maintain the MW and O2 affinity of

PolyHb solutions. Reaction parameters including type and concentration of cross-linking reagent, solution pH, temperature, reaction time, initial concentration of Hb and reducing enzymes and molar ratio of Hb to enzymes need to be optimized in order to obtain cross- link between Hb and the enzymes while maintaining antioxidant activity.

5.2.2 Scale-up of polymerization process

A standard blood transfusion unit in clinical use contains 450~500 mL of RBCs in storage solution [322] with Hb concentration ranging between 14~15 g/dL or 180~200 mL of packed RBCs [323]. However, the polymerization process described in this work can only prepare about 100 mL of PolyHb at a final concentration of 10~12 g/dL. Thus, in order to satisfy the Hb concentration required for clinical transfusion, a large scale polymerization process for bHb must be developed.

Reactor scale-up should ensure efficient gas-liquid exchange, which is related to the ratio of liquid surface area to liquid volume. For the preparation of L-PolyHb solutions, the gas-liquid exchange area can affect the effect the rate of Hb deoxygenation. Excessive amounts of Na2S2O4 would have to be used to quench excess O2 in the Hb solution if the argon purging failed to reduce the aqueous pO2 to below 15 mm Hg, which could elicit the formation of peroxides [324] and react with free glutaraldehyde [225], hereby reducing the MW of the PolyHb products.

131 5.2.3 Carbon monoxide saturated PolyHb for use in transfusion medicine

Despite being highly toxic at high concentrations, carbon monoxide (CO) has been reported to dilate blood vessels [325-327] and is an anti-apoptosis agent [328, 329].

Cabrales et al. [330] reported that transfusion with CO-saturated RBCs into hamsters improved tissue viability after ischemia. Another group, Sakai et al. [331] reported less liver oxidative damage after transfusion of CO-saturated Hb vesicles. Although the exact mechanisms underlying the anti-apoptosis and cyto-protective effects is still under debate, it is reasonable to predict that CO-PolyHb also can serve as an effective CO-carrier to be used in ischemic conditions or during hemorrhagic shock. In addition, CO-PolyHb solutions can be stored longer than deoxygenated PolyHb solutions. Thus, further research should be conducted to investigate the transfusion potential of CO-PolyHb solutions.

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