Design and characterization of novel albumin-based oxygen carriers as an alternative to whole blood

Abstract Introduction: Whole blood (WB) transfusion is routinely exercised for resuscitation following hemorrhage, supplementation in anemic cancer patients, and in perioperative blood management. Despite approximately 13.6 million units being transfused annually in the United States, supply of WB becomes severely constrained in rural environments and mass casualty events, creating severe constraints in emergency care. While the positive outcomes of WB use in patients are irrefutable, inconsistencies of supply in blood banks, questionable safety of the blood donated, and concern of acute hemolytic transfusion reactions highlight the importance of developing non-donor derived oxygen carriers for transfusion. Prior research on non-donor derived oxygen carriers has been centered on perfluorocarbons (PFC) and recombinant hemoglobin-based oxygen carriers (HBOCs). While PFC and HBOCs can carry oxygen in circulation, many adverse effects have been reported in clinical trials, ranging from organ failure (e.g. kidney, spleen and liver), hypertension, heart attack, and even death. Many of these adverse effects appear to be rooted in the designs of PFC and HBOCs. Without a protective membrane as seen in red blood cells, cell-free HBOCs tend to disassemble, extravasate, and autoxidize. This can lead to shorter circulation time, vasoconstriction, oxidative stress, and osmotic imbalance. To address these challenges, we sought to design and develop a series of novel recombinant Albumin-Based Oxygen Carrier (ABOC) proteins by fusing albumin with monomeric globins; myoglobin-albumin (MyB-ABOC), neuroglobin-albumin (NgB-ABOC), cytoglobin-albumin (CyB-ABOC), and, as a control, hemoglobin-albumin (HbB-ABOC). This ABOC design harnesses the high O2 affinities and independent O2release of monomeric globin proteins with the blood volume expansion and protective properties of albumin. We hypothesize that our ABOCs will serve as a viable, non-donor derived blood substitute, capable of effective O2 loading and off-loading, and suitable for safe on-demand transfusion into anemic patients. Methods: MyB-ABOC, NgB-ABOC, CyB-ABOC and HbB-ABOC were engineered with recombinant technology and expressed in E. coli system. Purified ABOCs were validated by SDS-PAGE and Western blot (anti-HSA antibody). O2 binding capacities were measured via oxygen dissociation assay (ODA), using purified human hemoglobin (Hgb) as a control. All samples were diluted to 5 uM in HEPES buffer, plated in a clear 96 well plate, and let to oxygenate at ambient air and temperature for 30 minutes. Using a closed system plate reader, nitrogen gas was blown over the plate (flow 13 SCFH) for 2 hours (T120). Optical density (OD) was measured from 375-577 nm wavelengths spanning Soret and Q-bands. Simple linear regression analysis was performed on percent total oxygenated protein from T0 to 120. Wilcoxon test was conducted on spectral OD measures from T0 to T120 (significance = P<0.05). Results: SDS-PAGE gel showed ABOC proteins at 73.7-78.5 kDa which corresponded to expected the size and further validated by Western blot. ODA demonstrated ABOCs and Hgb exhibited statistically significant differences in oxygen desaturation rates over time (F(4,95)=114.2, p < 0.0001). MyB-ABOC showed the fastest oxygen release (-0.189%/min), followed by Hgb (-0.131%/min), CyB-ABOC (-0.098%/min), HbB-ABOC (-0.087%/min), and NgB-ABOC (-0.039%/min). Over the course of 2 hours, ABOC signature Soret peak exhibited a red shift that suggests a reduction in bound O2. NgB-ABOC, CyB-ABOC, MyB-ABOC, and Hgb had significant differences in OD408-415 values from T0 to T120. Conclusion: These findings demonstrate ABOCs oxygen binding and release properties, with NgB-ABOC demonstrating highest O2 retention. The significant shifts in OD confirm ABOCs' capacity for O2 loading and off-loading in hypoxic environments comparable to severely anemic patients. The tissue-specific properties of the selected globin proteins contribute to varied O2 affinities, enabling future modulation of ABOC design. This novel ABOC design represents a promising advancement in developing antigen- and pathogen-free blood substitutes that could address critical supply shortages in emergency medicine, rural healthcare, and mass casualty scenarios, potentially revolutionizing transfusion medicine when WB is unavailable.