Oxygen therapeutics are being developed for a variety of applications in transfusion medicine. In order to reduce the side-effects (vasoconstriction, systemic hypertension, and oxidative tissue injury) associated with previous generations of oxygen therapeutics, new strategies are focused on increasing the molecular diameter of hemoglobin obtained from mammalian sources via polymerization and encapsulation. Another approach towards oxygen therapeutic design has centered on using naturally occurring large molecular diameter hemoglobins (i.e., erythrocruorins) derived from annelid sources. Therefore, the goal of Chapter 3 was to purify erythrocruorin from the terrestrial worm Lumbricus terrestris for oxygen therapeutic applications. Tangential flow filtration (TFF) was used as a scalable protein purification platform to obtain a >99% pure LtEc product, which was confirmed by size exclusion high performance liquid chromatography and SDS-PAGE analysis. In vitro characterization concluded that the ultra-pure LtEc product had oxygen equilibrium properties similar to human red blood cells, and a lower rate of auto-oxidation compared to human hemoglobin, both of which should enable efficient oxygen transport under physiological conditions. In vivo evaluation concluded that the ultra-pure product had positive effects on the microcirculation sustaining functional capillary density compared to a less pure product (~86% purity). In summary, we purified an LtEc product with favorable biophysical properties that performed well in an animal model using a reliable and scalable purification platform to eliminate undesirable proteins.
The long-term storage stability and portability of hemoglobin (Hb)-based oxygen carriers are important design criteria in the development of these therapeutics for use in emergency medicine in austere environments. Lyophilization or storing proteins in a freeze-dried form is known to increase storage lifetime and reduce overall weight. In Chapter 4, we lyophilized the extracellular mega-hemoglobin of the annelid Lumbricus terrestris and tested the storage stability at different temperatures and oxygenation conditions. Storage in refrigerated conditions for over 6 months in the presence of N2 reduced oxidation by 50%, while storage at room temperature in the presence of N2 reduced oxidation by 60%, all while maintaining the structural stability of the mega-hemoglobin. We also demonstrated a reliable strategy to freeze dry Hb in the presence of a minimally non-reducing disaccharide sugar that could be easily re-solubilized in deionized water.
Prior research has shown that Ecs possess slower auto-oxidation rates at physiological temperature than hHb, thus making them attractive candidates for use as RBC substitutes. Unfortunately, it was also observed that Ecs have a much lower circulatory half-life in vivo compared to other HBOCs. Hence, conjugating polyethylene glycol (PEG) to the surface of Ec was proposed as a simple strategy to increase Ec circulatory half-life. Therefore, in order to inform future in vivo studies with PEGylated Ec, we decided to investigate the structural stability and biophysical properties of variable PEG surface coverage on Ec compared to native Ec in Chapter 5. We observed an increase in PEG-Ec diameter and molecular weight and changes to the quaternary structure, secondary structure, and surface hydrophobicity after PEGylation. There was also an increase in oxygen binding affinity, oxygen offloading rate, and auto-oxidation rate for increasing PEGylation ratios. Weak dissociation of Ec was also observed after dense PEGylation caused by steric repulsion of the conjugated PEG chains. Hence, we determined an optimum Ec PEGylation ratio that resulted in a substantial size and MW increase along with preservation of oxygen binding properties.
In order to investigate other biocompatible surface coatings for Ec, we focused on surface coating LtEc with oxidized dextran (Odex), an inexpensive, biocompatible polysaccharide, to overcome the potential immune response upon transfusion in Chapter 6. Dextran was chemically modified via periodate-mediated ring opening oxidation to generate reactive aldehyde moieties then reacted with LtEc to yield LtEc surface conjugated with Odex (Odex-LtEc). Removal of unreacted species and purification of Odex-LtEc was accomplished via tangential flow filtration (TFF). Compared to native LtEc, Odex-LtEc displayed increased size and molecular weight. Odex-LtEc also displayed higher O2 affinity, slower O2 offloading rate, and higher auto-oxidation rate compared to native LtEc. Although certain HBOC characteristics such as increased size are linked to reduced oxidative tissue injury and vasoactivity, in vivo evaluation will be necessary to determine the safety and efficacy of Odex-LtEc as an RBC substitute.
Polydopamine (PDA) is a hydrophilic, biocompatible, bioinspired polymer coating used for biomedical nanoparticle assemblies and coatings and has previously been investigated for the surface-coating of hHb. PDA is typically synthesized via the self-polymerization of dopamine (DA) under alkaline (pH >8.0) conditions. However, at pH >8.0, the oligomeric structure of LtEc begins to dissociate. Therefore, in Chapter 7, we investigated a photocatalytic method of PDA polymerization on the surface of LtEc 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 in order to preserve the size and structure of LtEc. The resulting structural, biophysical, and antioxidant properties of PDA surface coated LtEc (PDA-LtEc) was characterized using various techniques. PDA-LtEc showed an increase in measured particle size, molecular weight, and surface zeta-potential with increasing reaction time from t = 2 hours to t = 16 hours compared to unmodified LtEc. PDA-LtEc reacted for 16 hours was found to have reduced oxygen-binding cooperativity and slower deoxygenation kinetics compared to PDA-LtEc with lower levels of polymerization (t = 2 hours). 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 (ferric iron reduction and free radical scavenging) when synthesized at a reaction time of t = 16 hours compared to LtEc. These antioxidant properties may prove beneficial for 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.