Structural Stability and Biophysical Properties of the Mega-Protein Erythrocruorin Are Regulated by Polyethylene Glycol Surface Coverage

Abstract

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.

Tangential flow filtration facilitated fractionation and PEGylation of low and high‐molecular weight polymerized hemoglobins and their biophysical properties

“…Conjugation of PEG on the surface of Hb (PEGylation of Hb) is a simple strategy that can reduce the vasoactivity of Hb (Vandegriff et al, 2003), prevent tissue extravasation and the associated side‐effects of cell‐free Hb (Cabrales & Friedman, 2007), and increase circulatory half‐life compared to Hb (Savla & Palmer, 2021). In addition, PEGylated Hbs (PEG‐Hbs) also exhibited high colloidal osmotic pressure (COP), which imparts favorable plasma expansion properties when the material is transfused into the bloodstream (Vandegriff et al, 1997).…”

Scalable manufacturing platform for the production of PEGylated heme albumin

“…PEGylation of HSA was performed before and after incorporation of heme using thiol‐maleimide chemistry as described in the literature for other proteins (Pires et al, 2020 ; Savla & Palmer, 2021 ). For the product heme‐PEG‐HSA, HSA was incubated with ×2 molar excess of iminothiolane hydrochloride (Fisher Scientific) and ×10 molar excess mPEG‐maleimide (5000 Da) (Laysan Bio) at 4°C for 16 h. Unreacted components were removed via diafiltration using TFF HF modules with 50 kDa MWCO in PBS.…”

“…This strategy first increases the aqueous solubility of heme by binding it to a carrier protein (HSA) that itself is soluble in aqueous solution, and then subsequently further improves the colloidal stability of heme‐HSA by PEGylating it. Previously, our lab has stabilized various proteins such as apohemoglobin, and the annelid mega‐Hb erythrocruorin via PEG surface conjugation leading to the increased colloidal stability of the PEGylated molecule (Pires et al, 2020 ; Savla & Palmer, 2021 ). In this study, we used thiol‐maleimide chemistry to facilitate PEGylation of HSA.…”

Photocatalytic Synthesis of a Polydopamine-Coated Acellular Mega-Hemoglobin as a Potential Oxygen Therapeutic with Antioxidant Properties

“…Previously, our group has used both oxidized dextran (Odex) and poly(ethylene glycol) (PEG) to surface coat LtEc. 15 One bioinspired surface coating gaining interest in biomedical applications is the mussel-inspired polydopamine (PDA), a polymer synthesized by the self-polymerization of the neurotransmitter dopamine (DA). 16 PDA is a desirable surface coating due to its hydrophilic and biocompatible properties and has been investigated for biomedical applications from nanoscale protein coating to macroscale medical device coating.…”