(188ct) Mixtures of Tense and Relaxed State Polymerized Human Hemoglobin Regulate Oxygen Affinity and Tissue Construct Oxygenation

Purpose: In hepatic hollow fiber (HF) bioreactors, the O₂ tension sensed by cultured hepatocytes has an effect on cell phenotype, which dictates the extent and duration of replicated liver functions. Zonal heterogeneity in the liver sinusoid, which stems from O₂ dependent regional variations in hepatocyte function, results in a “glucosat” in the liver. This functionality is important in maintaining blood glucose levels during feeding and fasting periods. A variety of detoxification functions, which rely on sequential phase I and phase II metabolic enzymes, also requires proper zonation of these enzymes along the hepatic acinus. Thus, replicating the zonation observed in the liver sinusoid is vital in bioartificial liver design. Our group has synthesized variable molecular weight (MW) HBOCs with low and high O₂ affinities for use as RBC substitutes. These materials are based on glutaraldehyde polymerization of Hb in either the low O₂ affinity (i.e. tense (T)) or high O₂ affinity (i.e. relaxed (R)) quaternary state. In these studies, the T- or R-state PolyHbs either have low or high O₂ affinity, however in tissue-engineered constructs it may be desirable to tune the O₂ affinity of the PolyHb solution to facilitate targeted O₂ delivery based on varying oxygenation requirements of tissues.

Methods: Human Hb (hHb) was purified via tangential flow filtration. Deoxygenated and oxygenated hHb were polymerized with glutaraldehyde to yield T- and R-state PolyhHb, respectively. Small hHb polymers, reduced glutaraldehyde, and excess quenching reagents were removed from the synthesized PolyhHb solutions using diafiltration. Stock solutions of T-state PolyhHb and R-state PolyhHb having the same molar concentration (on a heme basis) were prepared. These stock solutions were mixed at different molar ratios to yield mixtures of T- and R-state PolyhHbs. Hydrodynamic, heme concentration, total protein concentration, met-hemoglobin concentration, O₂-hHb/PolyhHb equilibrium curves, and gaseous ligand binding/release kinetics were each measured. To assess the ability of the PolyhHb mixtures to oxygenate tissue engineered constructs, we developed a computational model of a single hollow fiber (HF) in a HF bioreactor housing hepatocytes (i.e. bio-artificial liver assist device), where the inlet partial pressure of O₂ (pO₂), mixture fraction, and total PolyhHb concentration were varied to assess oxygenation within the device.

Results: The diameter of T- and R-state PolyhHbs was significantly larger than the diameter reported in the literature for cell-free hHb. Unlike hHb, the shape of the equilibrium O₂ binding curves obtained for PolyhHbs are not sigmoidal. This indicates a significant loss in cooperative binding of O₂ to Hb in PolyhHbs compared to unmodified hHb. As expected, the T-state PolyhHb exhibited lower O₂ affinity compared to hHb. In contrast, high O₂ affinity was observed for R-state PolyhHb compared to hHb and T-state PolyhHb. We observed that the P₅₀ for various mixtures of T- and R-state PolyhHbs were proportional to the molar ratio of pure T-state and pure R-state PolyhHb. Because of the extensive glutaraldehyde mediated intramolecular crosslinking, both T-state and R-state PolyhHbs display lower cooperativity (n) compared to unmodified hHb. for T-state PolyhHb was significantly higher than that obtained for 30:1 R-state PolyhHb and unmodified Hb. We observed that the rate of O₂ binding to T-state PolyhHb was significantly lower than unmodified hHb because of incomplete O₂ binding to T-state PolyhHb even under one atm of pure O₂. Polymerization of Hb in the T-state limits heme pocket accessibility to CO. Moreover, polymerization of Hb in the R-state results in more open conformation and greater heme pocket accessibility. In the computational model at low pO_2,ins, unmodified hHb was able to deliver more O₂ than the PolyhHbs synthesized in this study. This phenomenon likely results from the low cooperativity and high MW (i.e. lower diffusivity) of the synthesized PolyhHbs. As anticipated, T-state PolyhHb has the potential to oxygenate a HF bioreactor better than R-state PolyhHb and unmodified hHb. PolyhHb mixtures with T-state mole fractions greater than 50% resulted in less hypoxic and hyperoxic zones. Under these conditions, the ratio of the pericentral volume to the perivenous volume in the ECS doubled as the T-state mole fraction increased from 50 to 100%.

Conclusions: Molar mixtures of T-state and R-state PolyhHbs can yield HBOCs with tunable O₂ affinities. Additionally, O₂ transport simulations performed in this study suggest that mixtures of PolyhHbs with T-state molar fractions greater than 50% are best suited for hepatic HF bioreactor oxygenation.