Hemoglobin encapsulation in vesicles retards NO and CO binding and O₂release when perfused through narrow gas-permeable tubes

Abstract

Sakai H, Okuda N, Sato A, Yamaue T, Takeoka S, Tsuchida E. Hemoglobin encapsulation in vesicles retards NO and CO binding and O2 release when perfused through narrow gas-permeable tubes. Am J Physiol Heart Circ Physiol 298: H956 -H965, 2010. First published December 31, 2009 doi:10.1152/ajpheart.00741.2009.-Intravenous administration of cell-free Hb induces vasoconstriction and circulatory disorders, presumably because of the intrinsic affinities to endogenous nitric oxide (NO) and carbon monoxide (CO) as vasorelaxation factors and because of the facilitated O2 release that might induce autoregulatory vasoconstriction. We examined these gas reactions when Hb-containing solutions of four kinds were perfused through artificial narrow tubes at a practical Hb concentration (10 g/dl). Purified Hb solution, polymerized bovine Hb (Poly BHb), encapsulated Hb [Hb-vesicles (HbV), 279 nm], and red blood cells (RBCs) were perfused through a gas-permeable narrow tube (25 m inner diameter) at 1 mm/s centerline velocity. The level of reactions was determined microscopically based on the visible-light absorption spectrum of Hb. When the tube was immersed in NO and CO atmospheres, both NO binding and CO binding of deoxygenated Hb (deoxy-Hb) and PolyBHb in the tube was faster than those of HbV and RBCs, and HbV and RBCs showed almost identical binding rates. When the tube was immersed in a N2 atmosphere, oxygenated Hb and PolyBHb showed much faster O 2 release than did HbV and RBCs. PolyBHb showed a faster reaction than Hb because of the lower O2 affinity of PolyBHb than Hb. The diffusion process of the particles was simulated using Navier-Stokes and Maxwell-Stefan equations. Results clarified that small Hb (6 nm) diffuses laterally and mixes rapidly. However, the large-dimension HbV shows no such rapid diffusion. The purely physicochemical differences in diffusivity of the particles and the resulting reactivity with gas molecules are one factor inducing biological vasoconstriction of Hb-based oxygen carriers. microcirculation; blood substitutes; gas biology; liposome; erythrocytes CELL-FREE, HEMOGLOBIN-BASED oxygen carriers (HBOCs) have been developed for use as transfusion alternatives. Some examples are intramolecular cross-linked Hb, polymerized Hb, and polyethylene glycol conjugated Hbs (5). The realization of HBOCs has long been anticipated, because they are free of pathogens and blood-type antigens and are storable for a long time for using at emergency situations. Some are in the final stage of clinical trials (23). The major remaining hurdle before clinical approval of this earliest generation of HBOCs is vasoconstriction and resulting hypertension, which are presumably attributable to the high reactivity of Hb with endothelium-derived nitric oxide (NO) (26,28,55). It has been suggested that small molecular Hbs permeate across the endothelial cell layer to the space near by the smooth muscle and inactivate NO. However, cellular Hb-vesicles (HbV) that encapsulate concentrated Hb solution in phospholipid vesicles (37) ind...

Simple Method for Preparing Poly(ethylene glycol)-Surface-Conjugated Liposome-Encapsulated Hemoglobins: Physicochemical Properties, Long-Term Storage Stability, and Their Reactions with O2, CO, and NO

“…The k off , O2 values for acellular Hbs are comparable to most of the other studies reported in the literature (Table 1). However, Tuschida and coworkers 46 have reported higher values of k off , O2 for the Hb used in their study (84.00 s −1 ) as compared to values in the literature (≈40.00 s −1 ). 47 This higher value could be due to the use of organic solvents during the Hb purification process or heat pasteurization of the Hb used in their studies, which may have chemically modified the Hb and resulted in an increase in k off , O2 .…”

Encapsulation of hemoglobin inside liposomes surface conjugated with poly(ethylene glycol) attenuates their reactions with gaseous ligands and regulates nitric oxide dependent vasodilation

“…Moreover, significant retardation in O 2 offloading has been observed in RBCs when compared with acellular Hb (2–17 s −1 compared with 36–43 s −1 ) 28, 40, 41. In addition, PEG‐LEH dispersions had significantly lower k off , O2 ( P < 0.05) compared with other acellular HBOCs (17–23 s −1 compared with 22–130 s −1 ) 42–46. All these results suggest that encapsulation of Hb in a membrane bound particle similar to that of a RBC, plays a critical role in regulating the delivery of O 2 .…”

Cardiopulmonary hemodynamic responses to the small injection of hemoglobin vesicles (artificial oxygen carriers) in miniature pigs

“…Safety and efficacy evaluations of its use, mainly using rodents, as a resuscitative fluid for hemorrhagic shock are conducted energetically, 7,8 as a priming fluid for extracorporeal membrane oxygenator, 9 and as a targeted oxygen carrier for ischemic tissues [10][11][12] and tumors. 13 Actually, Hb encapsulation shields the toxic effects of stroma-free Hbs, especially preventing unwanted reactions of Hb with endogenously produced NO, 14,15 thereby preventing NO-related vasoconstriction and hypertension. 16 However, it is important to confirm the biocompatibility of ''vesicles.''…”