Previously, our lab developed high molecular weight (MW) tense (T) quaternary state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this study, we prepared PolybHb in the T and relaxed (R) quaternary state with ultrahigh MW (>500 kDa) with varying cross‐link densities, and investigated the effect of MW on key biophysical properties (i.e., O2 affinity, cooperativity (Hill) coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand‐binding kinetics, auto‐oxidation, and haptoglobin [Hp]‐binding kinetics). To further optimize current PolybHb synthesis and purification protocols, we performed a comprehensive meta‐data analysis to evaluate correlations between procedural parameters (i.e., cross‐linker:bovine hemoglobin (bHb) molar ratio, gas‐liquid exchange time, temperature during sodium dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T‐ and R‐state PolybHbs. Our results showed that, the duration of the fast‐step auto‐oxidation phase of R‐state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T‐state PolybHbs exhibited significantly higher bimolecular rate constants for binding to Hp and unimolecular O2 offloading rate constants compared to R‐state PolybHbs. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHbs. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.

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 (Hb) obtained from mammalian sources via polymerization and encapsulation. Another approach toward oxygen therapeutic design has centered on using naturally occurring large molecular diameter Hbs [i.e., erythrocruorins (Ecs)] derived from annelid sources. Therefore, the goal of this study was to purify Ec from the terrestrial worm Lumbricus terrestris for diverse oxygen therapeutic applications. Tangential flow filtration was used as a scalable protein purification platform to obtain a >99% pure Lumbricus terrestris Ec (LtEc) product, which was confirmed by size exclusion high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In vitro characterization concluded that the ultrapure LtEc product had oxygen equilibrium properties similar to those of human red blood cells and a lower rate of auto-oxidation compared to human Hb, both of which should enable efficient oxygen transport under physiological conditions. In vivo evaluation concluded that the ultrapure 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.

Photodynamic therapy (PDT) has been shown to effectively treat cancer by producing cytotoxic reactive oxygen species via excitation of photosensitizer (PS). However, most PS lack tumor cell specificity, possess poor aqueous solubility, and cause systemic photosensitivity. Removing heme from hemoglobin (Hb) yields an apoprotein called apohemoglobin (apoHb) with a vacant heme-binding pocket that can efficiently bind to hydrophobic molecules such as PS. In this study, the PS aluminum phthalocyanine (Al-PC) was bound to the apoHb-haptoglobin (apoHb-Hp) protein complex, forming an apoHb-Al-PC-Hp (APH) complex. The reaction of Al-PC with apoHb prevented Al-PC aggregation in aqueous solution, retaining the characteristic spectral properties of Al-PC. The stability of apoHb-Al-PC was enhanced via binding with Hp to form the APH complex, which allowed for repeated Al-PC additions to maximize Al-PC encapsulation. The final APH product had 65% of the active heme-binding sites of apoHb bound to Al-PC and a hydrodynamic diameter of 18 nm that could potentially reduce extravasation of the molecule through the blood vessel wall and prevent kidney accumulation of Al-PC. Furthermore, more than 80% of APH’s absorbance spectra were retained when incubated for over a day in plasma at 37 °C. Heme displacement assays confirmed that Al-PC was bound within the heme-binding pocket of apoHb and binding specificity was demonstrated by ineffective Al-PC binding to human serum albumin, Hp, or Hb. In vitro studies confirmed enhanced singlet oxygen generation of APH over Al-PC in aqueous solution and demonstrated effective PDT on human and murine cancer cells. Taken together, this study provides a method to produce APH for enhanced PDT via improved PS solubility and potential targeted therapy via uptake by CD163+ macrophages and monocytes in the tumor (i.e., tumor-associated macrophages). Moreover, this scalable method for site-specific encapsulation of Al-PC into apoHb and apoHb-Hp may be used for other hydrophobic therapeutic agents.

A wide variety of hemoglobin-based oxygen carriers (HBOCs) have been designed for use as red blood cell (RBC) substitutes in transfusion medicine, ex vivo organ perfusion, oxygen delivery to hypoxic tissues, and a myriad of other applications. However, hemoglobin (Hb) derived from annelids (erythrocruorins [Ecs]) comprise a natural class of HBOC, since they are larger in size (30 nm in diameter) and contain more heme groups per molecule (144 heme groups) compared to human Hb (hHb; 5 nm in diameter and 4 heme groups). The larger size of Ec compared to hHb reduces tissue extravasation from the vascular space, thus, reducing vasoconstriction, systemic hypertension, and tissue oxidative injury when used as an RBC substitute. In addition, prior research has shown that Ecs possess slower auto-oxidation rates than hHb at physiological temperature, 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. We observed an increase in PEG-Ec diameter and molecular weight (MW) and changes to the quaternary structure, secondary structure, and surface hydrophobicity after PEGylation. There was also an increase in oxygen binding affinity, reduction in oxygen offloading rate, and increase in 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 future studies, these materials will be tested in animal models to evaluate pharmacodynamics, pharmacokinetics, tissue oxygenation, microcirculatory responses, and overall safety.

Apohemoglobin (apoHb) contains vacant hydrophobic heme-binding pockets that can bind to a variety of hydrophobic molecules. Thus, apoHb is a promising protein for drug delivery, bioimaging, and heme scavenging. Unfortunately, apoHb has a short half-life and precipitates at physiological temperature. In this study, apoHb was surface-conjugated with poly(ethylene glycol) (PEG) to improve the therapeutic potential of apoHb. The scalable PEGylation process had >95% protein yield with ∼10 to 12 PEGs attached to each apoHb αβ dimer. The resulting PEG-apoHb had an average molecular weight of ∼80 to 90 kDa and a hydrodynamic diameter of 11 nm. PEG-apoHb maintained high heme-binding affinity and 30−40% of the hemebinding activity. Moreover, heme-bound and heme-free PEG-apoHb bound to haptoglobin, enabling PEG-apoHb to potentially target CD163+ macrophages and monocytes. Finally, PEG-apoHb was stable at physiological temperature with minimal precipitation. In summary, the in vitro results shown demonstrate that PEG-apoHb could be an effective in vivo heme scavenger during states of hemolysis.