Polymerized human cord hemoglobin assisted with ascorbic acid as a red blood cell substitute alleviating oxidative stress for blood transfusion

Abstract

Introduction: Blood transfusion is widely used in clinical settings, where considerable efforts have been devoted to develop red blood cell substitutes to overcome blood shortage and safety concerns. Among the several kinds of artificial oxygen carriers, hemoglobin-based oxygen carriers are promising due to their inherent good oxygen-binding and -loading properties. However, difficulties in prone to oxidation, production of oxidative stress, and injury in organs limited their clinical utility. In this work, we report a red blood cell substitute composed of polymerized human cord hemoglobin (PolyCHb) assisted with ascorbic acid (AA) that alleviates oxidative stress for blood transfusion.Methods: In this study, the in vitro impacts of AA on the PolyCHb were evaluated by testing the circular dichroism, methemoglobin (MetHb) contents and oxygen binding affinity before and after the addition of AA. In the in vivo study, guinea pigs were subjected to a 50% exchange transfusion with PolyCHb and AA co-administration, followed by the collection of blood, urine, and kidney samples. The hemoglobin contents of the urine samples were analyzed, and histopathologic changes, lipid peroxidation, DNA peroxidation, and heme catabolic markers in the kidneys were evaluated.Results: After treating with AA, there was no effect on the secondary structure and oxygen binding affinity of the PolyCHb, while the MetHb content was kept at 55%, which was much lower than that without AA treating. Moreover, the reduction of PolyCHbFe3+ was significantly promoted, and the content of MetHb could be reduced from 100% to 51% within 3 h. In vivo study results showed that PolyCHb assisted with AA inhibited the formation of hemoglobinuria, upgraded the total antioxidant capacity and downgraded the superoxide dismutase activity of kidney tissue, and lowered the expression of biomarkers for oxidative stress, e.g., malondialdehyde (ET vs ET+AA: 4.03±0.26 μmol/mg vs 1.83±0.16 μmol/mg), 4-hydroxy-2-nonenal (ET vs ET+AA: 0.98±0.07 vs 0.57±0.04), 8-hydroxy 2 deoxyguanosine(ET vs ET+AA: 14.81±1.58 ng/ml vs 10.91±1.36 ng/ml), heme oxygenase 1 (ET vs ET+AA: 1.51±0.08 vs 1.18±0.05) and ferritin (ET vs ET+AA: 1.75±0.09 vs 1.32±0.04). The kidney histopathology results also demonstrated that kidney tissue damage was effectively alleviated.Conclusion: In conclusion, these comprehensive results provide evidence for the potential role of AA in controlling oxidative stress and organ injury in the kidneys induced by PolyCHb, and suggest that PolyCHb assisted with AA has promising application for blood transfusion.

Engineering Synthetic Erythrocytes as Next‐Generation Blood Substitutes

“…Ascorbic acid, for example, has been combined with Hb for oxidative stress relief. [82,197] Nanozymes such as Au and CeO 2 nanoparticles have also been incorporated in different HBOCs, both displaying catalase-and SOD-like activity upon contact with H 2 O 2 . [99,198] Albumin-coated MnO 2 nanoparticles have been used as multifunctional nanozymes, with roles in O 2 generation, antioxidation, and ROS scavenging.…”

“…Reproduced with permission. [ 82 ] Copyright 2023, The Authors. D) Hb encapsulated in a lipid vesicle (Hb‐V).…”

“…One strategy involved isolating Hb from human cord blood, removing stromal proteins through hypotonic hemolysis, cross‐linking it with glutaraldehyde to a molecular weight below 100 kDa, membrane‐filtering, and redispersing it in phosphate buffered saline (PBS) at 8.0 g L −1 . [ 82 ] A further addition of ascorbic acid produced an injectable oxygenating antioxidant solution (Figure 4B), capable of alleviating oxidative stress in the kidney of guinea pigs and reducing the levels of lipid and DNA peroxidation (Figure 4C). [ 82 ] Another cross‐linking strategy consisted in using α‐succinimidyl‐ω‐maleimide and dithiothreitol to form a Hb nanoparticle, later coated with albumin and loaded with catalase and SOD.…”

“…[ 82 ] A further addition of ascorbic acid produced an injectable oxygenating antioxidant solution (Figure 4B), capable of alleviating oxidative stress in the kidney of guinea pigs and reducing the levels of lipid and DNA peroxidation (Figure 4C). [ 82 ] Another cross‐linking strategy consisted in using α‐succinimidyl‐ω‐maleimide and dithiothreitol to form a Hb nanoparticle, later coated with albumin and loaded with catalase and SOD. [ 83 ] Interestingly, catalase demonstrated good stability in the matrix over time, unlike SOD, a smaller enzyme, which was released prematurely from the construct.…”

“…Malondialdehyde (MDA) indicates lipid peroxidation; 8-hydroxy 2 deoxyguanosine (8-OHdG) indicates DNA peroxidation. Reproduced with permission [82]. Copyright 2023, The Authors.…”