Reduction and suppression of methemoglobin loaded in the polymeric nanoparticles intended for blood substitutes

Oxidation of hemoglobin (Hb) to nonfunctional methemoglobin (metHb) is a main challenge for the fabrication of an ideal Hb-based blood substitute. In this study, a novel nonenzymatic reduction and suppression route, combined with a fast prereduction, optimized double emulsion preparation, and a second sustaining postreduction, was developed to control the metHb in Hb-loaded nanoparticles (HbP) with porous microstructure to a desirable level. In the prereduction, the metHb in the raw Hb was effectively reduced from over 90% to 1.2% using sodium dithionite following gel filtration separation. During the preparation, higher the emulsion strength performed, higher was the extent of Hb oxidized. PEGylated polymer and addition of miscible solvent, such as acetonitrile, into the oil phase could pronouncedly suppress metHb formation. The resultant metHb level in HbP under the optimal fabrication was about 5.6%, which could be further reduced to 1.4% by the model reducing agents in human plasma with the help of superoxide dismutatse and catalase system, which are capable of sustaining postreduction. The oxygen dissociation curve of the HbP was close to that of native Hb, indicating that the oxygen-carrying ability of the Hb, despite initially losing this function due to the severe oxidation, recovered and retained well. The results achieved are promising for the fabrication of blood substitutes with controlled metHb level, which can fulfill the binding/delivering oxygen to tissues in vivo for future trials.