Mixtures of tense and relaxed state polymerized human hemoglobin regulate oxygen affinity and tissue construct oxygenation.

Pure tense (T) and relaxed (R) quaternary state polymerized human hemoglobins (PolyhHbs) were synthesized and their biophysical properties characterized, along with mixtures of T- and R-state PolyhHbs. It was observed that the oxygen affinity of PolyhHb mixtures varied linearly with T-state mole fraction. Computational analysis of PolyhHb facilitated oxygenation of a single fiber in a hepatic hollow fiber (HF) bioreactor was performed to evaluate the oxygenation potential of T- and R-state PolyhHb mixtures. PolyhHb mixtures with T-state mole fractions greater than 50% resulted in hypoxic and hyperoxic zones occupying less than 5% of the total extra capillary space (ECS). 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%. These results show the effect of varying the T/R-state PolyhHb mole fraction on oxygenation of tissue-engineered constructs and their potential to oxygenate tissues.

Erythrocytes Are Oxygen-Sensing Regulators of the Cerebral Microcirculation.

Energy production in the brain depends almost exclusively on oxidative metabolism. Neurons have small energy reserves and require a continuous supply of oxygen (O2). It is therefore not surprising that one of the hallmarks of normal brain function is the tight coupling between cerebral blood flow and neuronal activity. Since capillaries are embedded in the O2-consuming neuropil, we have here examined whether activity-dependent dips in O2 tension drive capillary hyperemia. In vivo analyses showed that transient dips in tissue O2 tension elicit capillary hyperemia. Ex vivo experiments revealed that red blood cells (RBCs) themselves act as O2 sensors that autonomously regulate their own deformability and thereby flow velocity through capillaries in response to physiological decreases in O2 tension. This observation has broad implications for understanding how local changes in blood flow are coupled to synaptic transmission.

Mathematical model of NO and O2 transport in an arteriole facilitated by hemoglobin based O2 carriers.

The increasing demand for donated human blood has spurred research to develop hemoglobin-based O(2) carriers (HBOCs) that can be used as red blood cell (RBC) substitutes. However, in vivo studies of acellular HBOCs have shown an increase in mean arterial pressure following transfusion that has been attributed to the HBOC's ability to scavenge NO (an important vasodilator that is synthesized by endothelial cells in the blood vessel wall that signals neighboring smooth muscle cells to relax). In this study, a mathematical model was developed to describe NO and O(2) transport in an arteriole containing a mixture of acellular HBOCs and RBCs. The acellular HBOCs studied in this work possessed a wide range of O(2) affinities, O(2) dissociation rate constants and NO reactivities in order to evaluate their effect on O(2) tension and NO concentration in the arteriole tissue region. By focusing on the concentration of NO that is localized in the arteriole smooth muscle cell region, the model can predict the vasopressor response of HBOCs. The results of this study confirmed that acellular HBOCs scavenge large amounts of NO from the entire arteriole (approximately 50% or more NO compared to RBCs only). A recombinant Hb, rHb3011, displayed the least NO reactivity and consequently left the most NO remaining in the arteriole. The NO concentration in the arteriole with respect to the other HBOCs studied was proportional to their NO reactivity. Therefore, the results of this study demonstrate that NO scavenging is an unavoidable consequence of transfusing HBOCs. To prevent or reduce vasodilatation, we suggest administration of NO by either inhaling NO or transfusing nitrite into the blood stream followed by transfusion of HBOC.

Modern cross-linking strategies for synthesizing acellular hemoglobin-based oxygen carriers.

Unmodified cell-free hemoglobin (Hb) is structurally unstable when transfused into the blood stream (Valeri et al., Artif Cells Blood Substit Immobil Biotechnol. 2000;28:451-475; Chan et al., Toxicol Pathol. 2000;28:635-642; Eike, Dissertation, 2005; Eike and Palmer, Biotechnol Prog. 2004;20:946-952). This review examines some of the latest chemical strategies used over the last 5 years to intra- and intermolecularly cross-link Hb, thereby stabilizing its quaternary structure. Therefore, this work will address the following aspects: (1) site-specific chemical modifications of Hb and (2) non-site-specific chemical modifications of Hb, including, but not limited to, PolyHeme, Hemopure, Oxyglobin, and SOD-Hb. Current strategies for synthesizing PEGylated Hb is outside the scope of this review and will not be discussed herein. For a more thorough review of PEGylated Hb, the reader is directed to the following works: Cabrales and Friedman, Transfus Alternatives in Transfus Med. 2007;9:281-293 and Winslow, Biochim Biophys Acta, 2008;1784(10):1382-1386.

Liposome-encapsulated actin-hemoglobin (LEAcHb) artificial blood substitutes.

A new approach to enhance the circulation persistence of liposomes has been applied to develop liposome-encapsulated actin-hemoglobin (LEAcHb) dispersions as potential blood substitutes by introducing an actin matrix into the liposome aqueous core. Asymmetric flow field-flow fractionation coupled with multi-angle static light scattering was used to study the shape, size distribution, and encapsulation efficiency of liposome-encapsulated hemoglobin (LEHb) and LEAcHb dispersions. By polymerizing monomeric actin into filamentous actin inside the liposome aqueous core, LEAcHb particles transformed into a disk-like shape. We studied the effect of an encapsulated actin matrix on the size distribution, hemoglobin (Hb) encapsulation efficiency, oxygen affinity, and methemoglobin (MetHb) level of LEAcHb dispersions, and compared them with plain LEHb dispersions (without actin). LEHb, and LEAcHb dispersions extruded through 400 nm membranes were injected into rats and it was observed that LEAcHb dispersions with 1mg/mL of actin enhanced the circulatory half-life versus LEHb dispersions. The circulatory characteristics of empty PEGylated and non-PEGylated actin-containing liposomes (without Hb) were studied as controls for the LEHb and LEAcHb dispersions in this paper, which displayed maximum circulatory half-lives greater than 72 h. Taken together the results of this study supports our hypothesis that a lipid membrane supported by an underlying actin matrix will extend the circulatory half-life of LEHb dispersions.