Abstract: BACKGROUND: Molecular stability and biocompatibility of hemerythrin surpass those of human and mammalian hemoglobin, making it a potential candidate for a safer and more effective erythrocyte substitute after modification.
OBJECTIVE: To prepare multi-modified hemerythrin nanoparticles, characterize them, and test their performance in vitro. 
METHODS: The hemerythrin of Sipunculus sphenodontus was separated and purified by tangential flow ultrafiltration. The intramolecular cross-linking was completed by genipin. The nanoparticles were encapsulated by dopamine, and passivated by polyethylene glycol to obtain multi-modified hemerythrin nanoparticles. The physicochemical properties of the nanoparticles were characterized. Hemerythrin nanoparticles, hemerythrin, and hemoglobin oxygen carrier HBOC-201 with different mass concentrations (0, 0.25, 0.5, 1.0, and 2.0 mg/mL) were incubated with macrophages for 6 and 24 hours, and with endothelial cells for 24 hours. The cell survival rate was detected by CCK-8 assay. The levels of nitric oxide and vascular cell adhesion factor 1 in the culture medium of endothelial cells were detected by ELISA.
RESULTS AND CONCLUSION: (1) Under electron microscopy, hemerythrin nanoparticles were ellipsoidal, with a dense outer membrane and a relatively uniform internal texture. The particle size was (150.12±1.67) nm; the dispersion index was 0.21±0.03; the Zeta potential was (-24.54±2.61) mV; the half-saturated oxygen partial pressure was (0.97±0.15) kPa, and the Hill coefficient was 1.49±0.16. (2) After incubation for 6 hours, within the mass concentration range of ≤1.0 mg/mL, the survival rates of macrophages in the hemerythrin nanoparticle group, the hemerythrin group, and the HBOC-201 group were all above 85%. At a mass concentration of 2.0 mg/mL, only the survival rate of macrophages in the hemerythrin nanoparticle group was above 80%. After incubation for 24 hours, the survival rates of macrophages in the three groups were all lower than 80%, among which the survival rate of macrophages in the hemerythrin nanoparticle group was higher than that in the hemerythrin group and the HBOC-201 group (P < 0.05). (3) With the increase of drug concentration, the survival rate of vascular endothelial cells in the three groups decreased. At 1.0 mg/mL or 2.0 mg/mL mass concentration, the survival rate of cells in the hemerythrin nanoparticle group was higher than that in the hemerythrin group and HBOC-201 group (P < 0.05). At the same mass concentration, the nitric oxide level in the hemerythrin nanoparticle group was higher than that in the hemerythrin group and HBOC-201 group (P < 0.05). In the range of 0.25-2.0 mg/mL mass concentration, the vascular cell adhesion factor 1 level in the hemerythrin nanoparticle group was lower than that in the hemerythrin group and HBOC-201 group (P < 0.05). (4) The results showed that the hemerythrin nanoparticles modified with intramolecular cross-linking and polydopamine/polyethylene glycol had good oxygen-carrying activity in vitro, better anti-phagocytic performance, and less cytotoxicity.

Key words: erythrocyte substitutes, hemerythrin, chemical modification, cell experiments, hemoglobin-based oxygen carriers