Synthesis and physicochemical characterization of a series of hemoglobin-based oxygen carriers: Objective comparison between cellular and acellular types

A series of hemoglobin (Hb)-based O₂ carriers, acellular and cellular types, were synthesized and their physicochemical characteristics were compared. The acellular type includes intramolecularly cross-linked Hb (XLHb), polyoxyethylene (POE)-conjugated pyridoxalated Hb (POE-PLP-Hb), hydroxyethylstarch-conjugated Hb (HES-XLHb), and glutaraldehyde-polymerized XLHb (Poly-XLHb). The cellular type is Hb-vesicles (HbV) of which the surface is modified with POE (POE-HbV). Their particle diameters are 7 ± 2, 22 ± 2, 47 ± 17, 68 ± 24, and 224 ± 76 nm, respectively, thus all the materials penetrate across membrane filters with 0.4 μm pore size, though only the POE-HbV cannot penetrate across the filter with 0.2 μm pore size. These characteristics of permeability are important to consider an optimal particle size in microcirculation in vivo. POE-PLP-Hb ([Hb] = 5 g/dL) showed viscosity of 6.1 cP at 332 s^-1 and colloid osmotic pressure (COP) of 70.2 Torr, which are beyond the physiological conditions (human blood, viscosity = 3-4 cP, COP = ca. 25 Torr). XLHb and Poly-XLHb showed viscosities of 1.0 and 1.5 cp, respectively, which are significantly lower than that of blood. COP of POE-HbV is regulated to 20 Torr in 5% human serum albumin (HSA). HES-XLHb and POE-HbV/HSA showed comparable viscosity with human blood. Microscopic observation of human red blood cells (RBC) after mixing blood with POE-PLP-Hb or HES-XLHb disclosed aggregates of RBC, a kind of sludge, indicating a strong interaction with RBC, which is anticipated to modify peripheral blood flow in vivo. On the other hand, XLHb and POE-HbV showed no rouleaux or aggregates of RBC. The acellular Hbs (P₅₀ = 14-32 Torr) have their specific O₂ affinities determined by their structures, while that of the cellular POE-HbV is regulated by coencapsulating an appropriate amount of an allosteric effector (e.g., P₅₀ = 18, 32 Torr). These differences in physicochemical characteristics between the acellular and cellular types indicate the advantages of the cellular type from the physiological points of view.