Photocatalytic Synthesis of a Novel Polydopamine-Coated Acellular Mega-Hemoglobin As a Potential Oxygen Therapeutic

Hemoglobin-based oxygen carriers (HBOCs) are being developed to overcome limitations associated with transfusion of donated red blood cells (RBCs) such as transmission of blood borne pathogens and limited shelf-life. Annelid erythrocrorin (Ec) derived from the worm Lumbricus terrestris (Lt) is an acellular mega-hemoglobin that has promise as a potential HBOC due to the large size of its oligomeric structure, thus overcoming limitations of unmodified circulating cell-free hemoglobin (Hb). With a large molecular weight of 3.6 MDa compared to 64.5 kDa for human Hb (hHb) and 144 oxygen-binding globin subunits compared to the 4 globin subunits of hHb, LtEc does not extravasate from the circulation compared to hHb. LtEc is stable in circulation without RBC membrane encapsulation and has a lower rate of auto-oxidation compared to acellular hHb, which allows the protein to remain functional for longer periods of time in circulation. Surface coatings, such as poly-ethylene glycol (PEG) and oxidized-dextran (Odex), have been investigated to reduce the immune response and improve the circulation time of LtEc in vivo. Polydopamine (PDA) is a hydrophobic, biocompatible polymer coating used for biomedical nanoparticle assemblies and has previously been investigated for the surface-coating of hHb. PDA is typically synthesized via the self-polymerization of dopamine (DA) in alkaline (pH >8.0) conditions. At pH >8.0, the oligomeric structure of LtEc begins to dissociate. We investigated a photocatalytic method of PDA polymerization using 9-mesityl-10-methylacridinium tetrafluoroborate (Acr-Mes) to drive PDA polymerization under physiological conditions (pH 7.4, 25 ˚C) over 2, 5, and 16 hours to preserve the size and structure of LtEc. PDA-LtEc showed an increase in measured particle size via DLS, molecular weight via HPLC, and surface ζ-potential with increasing reaction time from t = 2h to t = 16h compared to unmodified LtEc. PDA-LtEc reacted for 16h was found to have reduced oxygen-binding cooperativity and deoxygenation kinetics compared to PDA-LtEc with lower levels of polymerization (t = 2h), but statistically significant differences in were not measured. The thickness of the PDA coating can be controlled and in turn the biophysical properties can be tuned by changing various reaction conditions. PDA-LtEc was shown to demonstrate an increased level of antioxidant capacity when synthesized with a reaction time of t = 16h. These antioxidant properties may prove beneficial to oxidative protection of PDA-LtEc during its time in the circulation. Hence, we believe that PDA-LtEc is a promising oxygen therapeutic for potential use in transfusion medicine applications.