Department of Bioengineering, University of California San Diego, La Jolla, California 92093, United States.
William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio 43210, United States.
ACS Appl Bio Mater. 2024 Aug 19;7(8):5188-5200. doi: 10.1021/acsabm.4c00273. Epub 2024 Jul 5.
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. (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).
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.
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.
由于镰状细胞贫血、疟疾和地中海贫血等慢性病的诊断增加，对红细胞（RBC）的需求不断上升。尽管进行了许多商业尝试，但美国食品和药物管理局（FDA）尚未批准用于人体的人工 RBC。现有的 RBC 替代品采用了各种策略来运输氧气、延长循环时间和减少器官毒性，但都没有复制 RBC 的天然保护机制，这种机制可以防止血红蛋白（Hb）二聚化和血红素铁氧化。（蚯蚓）血影蛋白（LtEc）是一种天然存在的细胞外血红蛋白（Hb），具有很大的应用前景：分子直径大（30nm）、分子量高（3.6MDa）、自氧化率低、一氧化氮清除能力有限。这些特性使 LtEc 成为 RBC 替代品的理想候选物。然而，LtEc 的半衰期短，这是一个显著的缺点。为了解决这个问题，我们探索了 LtEc 的巯基介导的表面聚乙二醇化（PEG-LtEc），PEG 表面覆盖率不同。PEG 表面覆盖率超过 40%会使 LtEc 不稳定，形成 LtEc 大小 1/12 的较小亚基。因此，我们评估了两种 PEG 表面覆盖率选项：PEG-LtEc-0.2（20%PEG 化）和 PEG-LtEc-1.0（100%PEG 化）。
我们使用背部窗口室和导管的金黄地鼠进行实验，以评估这些溶液的功效。我们测量微血管参数、器官功能、脑血流、循环时间、平均动脉压、心率和血气，并进行组织学检查以筛查毒性。
我们的研究结果表明，与 LtEc 相比，PEG-LtEc 分子在恢复微血管参数、器官功能、脑血流和循环时间方面都具有显著优势。值得注意的是，PEG-LtEc-1.0 显示出更好的微血管灌注效果，尽管其自氧化率高于 PEG-LtEc-0.2。这些结果突出了 PEG 化在组织灌注和器官健康方面的优势，同时也强调了其局限性。
