Increasing the stability of <i>Lumbricus terrestris</i> erythrocruorin <i>via</i> poly(acrylic acid) conjugation

<p>Since donated red blood cells must be constantly refrigerated, they are often unavailable in remote areas and battlefields. The goal of this study was to synthesize a highly stable blood substitute that does not require refrigeration. Specifically, the extracellular haemoglobin (a.k.a. erythrocruorin, Ec) of the earthworm <i>Lumbricus terrestris</i> erythrocruororin (LtEc) was cross-linked with poly(acrylic acid) (PAA) and ethylene diamine (EDA). PAGE analysis of the LtEc nanoparticles reveals cross-linking between subunits, while dynamic light scattering and scanning electron microscopy show that cross-linking significantly increases the size of the LtEc nanoparticles (164 ± 13.9 nm). Cross-linking also significantly increased the thermal stability of the LtEc nanoparticles by 10 °C (<i>T</i>_m = 72 ± 0.84 °C) relative to native LtEc (<i>T</i>_m = 62 ± 0.6 °C). In addition, while native LtEc rapidly dissociates at pH 9, the LtEc nanoparticles resist subunit dissociation up to pH 10. The oxygen affinity of the LtEc nanoparticles (P₅₀ = 6.85 ± 0.13 mm Hg) is much higher than native LtEc (P₅₀ = 26.67 ± 0.4 mm Hg), but the cooperativity (<i>n</i> = 2.43 ± 0.12) is not affected. Altogether, these results show that cross-linking LtEc with PAA and EDA provides a potential blood substitute with increased stability and oxygen affinity.</p