Degree of PEGylatation of Lumbricus terrestris Hemoglobin Improves Microcirculatory Blood Flow but Increases the Rate of Auto-Oxidation.

INTRODUCTION: The demand for red blood cells (RBCs) is on the rise due to the increasing diagnosis of chronic diseases such as sickle cell anemia, malaria, and thalassemia. Despite many commercial attempts, there are no U.S. FDA-approved artificial RBCs for use in humans. Existing RBC substitutes have employed various strategies to transport oxygen, extend the circulation time, and reduce organ toxicity, but none have replicated the natural protective mechanisms of RBCs, which prevent hemoglobin (Hb) dimerization and heme iron oxidation. Lumbricus terrestris (earthworm) erythrocruorin (LtEc) is a naturally occurring extracellular hemoglobin (Hb) with promising attributes: large molecular diameter (30 nm), high molecular weight (3.6 MDa), low auto-oxidation rate, and limited nitric oxide-scavenging properties. These characteristics make LtEc an ideal candidate as an RBC substitute. However, LtEc has a significant drawback, its short circulatory half-life. To address this issue, we explored thiol-mediated surface PEGylation of LtEc (PEG-LtEc) at varying polyethylene glycol (PEG) surface coverages. Increasing PEG surface coverage beyond 40% destabilizes LtEc into smaller subunits that are 1/12th the size of LtEc. Therefore, we evaluated two PEG surface coverage options: PEG-LtEc-0.2 (20% PEGylation) and PEG-LtEc-1.0 (100% PEGylation). METHODS: We conducted experiments using golden Syrian hamsters with dorsal window chambers and catheters to assess the efficacy of these solutions. We measured microvascular parameters, organ function, cerebral blood flow, circulation time, mean arterial pressure, heart rate, and blood gases and performed histology to screen for toxicity. CONCLUSION: Our findings indicate that both PEG-LtEc molecules offer significant benefits in restoring microvascular parameters, organ function, cerebral blood flow, and circulation time compared to LtEc alone. Notably, PEG-LtEc-1.0 showed superior microvascular perfusion, although it exhibited a higher rate of auto-oxidation compared to PEG-LtEc-0.2. These results underscore the advantages of PEGylation in terms of tissue perfusion and organ health while highlighting its limitations.

Toxic side-effects of diaspirin cross-linked human hemoglobin are attenuated by the apohemoglobin-haptoglobin complex.

Alpha-alpha diaspirin-crosslinked human hemoglobin (DCLHb or ααHb) was a promising early generation red blood cell (RBC) substitute. The DCLHb was developed through a collaborative effort between the United States Army and Baxter Healthcare. The core design feature underlying its development was chemical stabilization of the tetrameric structure of hemoglobin (Hb) to prevent Hb intravascular dimerization and extravasation. DCLHb was developed to resuscitate warfighters on the battlefield, who suffered from life-threatening blood loss. However, extensive research revealed toxic side effects associated with the use of DCLHb that contributed to high mortality rates in clinical trials. This study explores whether scavenging Hb and heme via the apohemoglobin-haptoglobin (apoHb-Hp) complex can reduce DCLHb associated toxicity. Awake Golden Syrian hamsters were equipped with a window chamber model to characterize the microcirculation. Each group was first infused with either Lactated Ringer's or apoHb-Hp followed by a hypovolemic infusion of 10% of the animal's blood volume of DCLHb. Our results indicated that animals pretreated with apoHb-Hb exhibited improved microhemodynamics vs the group pretreated with Lactated Ringer's. While systemic acute inflammation was observed regardless of the treatment group, apoHb-Hp pretreatment lessened those effects with a marked reduction in IL-6 levels in the heart and kidneys compared to the control group. Taken together, this study demonstrated that utilizing a Hb and heme scavenger protein complex significantly reduces the microvasculature effects of ααHb, paving the way for improved HBOC formulations. Future apoHb-Hp dose optimization studies may identify a dose that can completely neutralize DCLHb toxicity.

A Challenge for Engineering Biomimetic Microvascular Models: How do we Incorporate the Physiology?

The gap between in vitro and in vivo assays has inspired biomimetic model development. Tissue engineered models that attempt to mimic the complexity of microvascular networks have emerged as tools for investigating cell-cell and cell-environment interactions that may be not easily viewed in vivo. A key challenge in model development, however, is determining how to recreate the multi-cell/system functional complexity of a real network environment that integrates endothelial cells, smooth muscle cells, vascular pericytes, lymphatics, nerves, fluid flow, extracellular matrix, and inflammatory cells. The objective of this mini-review is to overview the recent evolution of popular biomimetic modeling approaches for investigating microvascular dynamics. A specific focus will highlight the engineering design requirements needed to match physiological function and the potential for top-down tissue culture methods that maintain complexity. Overall, examples of physiological validation, basic science discoveries, and therapeutic evaluation studies will emphasize the value of tissue culture models and biomimetic model development approaches that fill the gap between in vitro and in vivo assays and guide how vascular biologists and physiologists might think about the microcirculation.