Core–Shell Structured Hemoglobin Nanoparticles as Artificial O₂ Carriers

Abstract

This paper describes the synthesis and O 2 binding properties of core− shell structured hemoglobin (Hb) nanoparticles (NPs), artificial O 2 carriers of five types, as designed for use as red blood cell (RBC) substitutes. Human adult Hbs were polymerized using α-succinimidyl-ω-maleimide and dithiothreitol in spheroidal shapes to create parent particles. Subsequent covalent wrapping of the sphere with human serum albumin (HSA) yielded 100 nm-diameter Hb nanoparticles (HbNPs). The HbNP showed higher O 2 affinity than that of RBC, but NPs prepared under a N 2 atmosphere exhibited low O 2 affinity. Entirely synthetic particles comprising recombinant human adult Hb and recombinant HSA were also fabricated. Using a recombinant Hb (rHb) variant in which Leu-β28 of the heme pocket had been replaced with Phe, we found somewhat low O 2 affinity of rHb(βL28F)NP. Particles made of stroma-free Hb (SFHb) containing natural antioxidant enzyme catalase (SFHbNP) formed a very stable O 2 complex, even in aqueous H 2 O 2 solution. The SFHbNP showed good blood compatibility and did not affect the blood cell component functionality. The circulation half-life of SFHbNP in rats was considerably longer than that of naked Hb. All results indicate these Hb-based NPs as useful alternative materials for RBC and as a useful O 2 therapeutic reagent in diverse medical scenarios.

Poly(2-ethyl-2-oxazoline)-Conjugated Hemoglobins as a Red Blood Cell Substitute

“…These values indicated somewhat higher O 2 affinities than Hb (13 Torr, n = 2.5) and HbA (12 Torr, n = 2.4). We described earlier that the chemical modifications of cysteine-β93 and surface lysine residues of Hb by SMP interfered with the quaternary structure transition from the relaxed (R) state to tense (T) state, which caused a marked enhancement of O 2 affinity. , The high O 2 affinity and low cooperativity observed for POx-Hb and POx-HbA were derived from the same mechanism. This interpretation is consistent with the findings reported by Vandegriff et al, Kluger et al, Manjula et al, Li et al, and Styslinger et al The higher O 2 affinity is expected to exert an excellent O 2 delivery to hypoxic tissues, such as in hemorrhagic shock states.…”

“…47 As a core protein, we used porcine Hb, as obtained from porcine RBCs of specific pathogen-free (SPF) swine using the procedures that we reported earlier. 29,49 A cross-linker, N-succinimidyl 3-maleimi- dopropionate (SMP), was reacted with Hb surface lysines to yield maleimido-activated Hb (MA-Hb). Subsequently, a sulfhydryl group of POx-SH was coupled site-specifically to the maleimide terminuses of MA-Hb.…”

“…A unique sulfhydryl-group-terminated POx, POx-SH, was synthesized by the coupling of POx (Mw: 5 kDa) with 3,3′-dithiodipropionic acid followed by reduction of the middle disulfide bond (Figure A) . As a core protein, we used porcine Hb, as obtained from porcine RBCs of specific pathogen-free (SPF) swine using the procedures that we reported earlier. , A cross-linker, N -succinimidyl 3-maleimidopropionate (SMP), was reacted with Hb surface lysines to yield maleimido-activated Hb (MA-Hb). Subsequently, a sulfhydryl group of POx-SH was coupled site-specifically to the maleimide terminuses of MA-Hb.…”

“…Synthesis of POx-SH and preparation of purified Hb and HbA were described in our earlier reports. ,, A freshly prepared DMSO solution of SMP (170 mM, 3 mL) was added dropwise into a PBS solution of carbonyl Hb (1 mM, 30 mL) in a round-bottom flask (100 mL volume). The mixture was stirred for 1 h at 25 °C under CO. After removing unreacted excess SMP using a gel filtration chromatography (GFC) column (Sephadex G-25 Superfine; Cytiva, the running buffer; PBS), the volume of the obtained maleimide-activated Hb (MA-Hb) was adjusted to 90 mL ([Hb] = 0.33 mM) by adding PBS.…”

“…Recently, decreased numbers of donors attributable to declining birthrates and aging populations have become a social difficulty worldwide. − In 2020, the COVID-19 pandemic caused a severe blood shortage, which disrupted general medical care provision in hospitals. , For that reason, international organizations must secure artificial blood, particularly viable red blood cell (RBC) substitutes . During the past few decades, hemoglobin (Hb)-based O 2 carriers (HBOCs) of various kinds have been manufactured and evaluated, − such as intramolecular cross-linked Hbs, − polymerized Hbs, − poly­(ethylene glycol)-conjugated Hbs (PEG-Hbs), − oligosaccharide-conjugated Hbs, Hb nano/microparticles, − and liposome–encapsulated Hbs. − In fact, HBOCs offer several advantages over blood transfusion, which include their capability of long-term storage, their lack of infection risk, and their obviation of blood-type preparation and testing. Simple and cost-effective PEG-Hbs have been investigated widely as promising materials.…”