Engineering a novel biomaterial for oxygen transport applications

摘要 基于血红蛋白（Hb）的氧（O2）载体（HBOCs）目前正被开发为红细胞 (RBC)用于输血医学的替代品。尽管有重大的商业发展， 聚合血红蛋白（PolyHb）溶液（即HemopureTM（OPKBiotech， 剑桥，MA），戊二醛聚合的牛Hb;和PolyHeme β（Northfield Laboratories Inc.， Evanston，IL），戊二醛聚合的吡啶氧基化的人Hb）阻碍进一步发育。两 这些商业产品引起微循环水平的血管收缩，并导致 全身性高血压和氧化性组织损伤。这些副作用被假设为通过以下方式发生： 一氧化氮（NO）清除或氧（O2）过量供应机制，并都加剧了聚血红蛋白 外渗到组织间隙中。根据这两种潜在机制，显然PolyHb大小将 对血管收缩、全身性高血压和氧化组织毒性的程度有深远的影响。 然而，商业PolyHb产品是具有宽尺寸分布的复杂混合物，仅由以下定义： 在其制造中使用的超滤膜的尺寸截止。此外，这些混合物是 已知含有高达1%的单个四聚体Hb分子和显著更高比例的低聚体Hb分子。 分子量（MW）Hb低聚物（80%，MW < 500 kDa）。因此，观察到的副作用 临床/临床前试验归因于具有不同大小和点的低MW Hb聚合物的混合物， 化学修饰，而不是任何一个单一的PolyHb分子。这就排除了对 这些复杂的PolyHb混合物的各个成分如何与血管系统相互作用。 因此，一个重要的进步是能够产生分子均匀，单分散， 高MW PolyHb纳米结构。在本申请中，我们假设分子直径和 重组PolyHb（rPolyHb）的拓扑结构将调节血管活性和对组织的氧化损伤。到 为了验证我们的假设，我们提出以下具体目标： 具体目标1：使用正交分裂剪接内含肽来产生定义明确的、单分散的、高分子量的内含肽 rPolyHb纳米结构。 具体目标2a：分析内皮功能在血管活性和氧化应激发生中的作用。 不同大小的rPolyHb的组织损伤。 具体目的2b：评价rPolyHbs在正常豚鼠和HFSD豚鼠中的药代动力学 猪 具体目标3：评价rPolyHbs恢复组织氧合和优化生存的能力， 严重失血 所提出的工作既有意义又有创新性，因为它寻求开发安全有效的 rPolyHbs用于输血医学。此外，最先进的生物物理技术和两种独特的 将使用动物模型来了解rPolyHb生理反应并确定临床潜力 这些新材料。

Abstract Hemoglobin (Hb)-based oxygen (O2) carriers (HBOCs) are currently being developed as red blood cell (RBC) substitutes for use in transfusion medicine. Despite significant commercial development, recent late stage clinical results of polymerized hemoglobin (PolyHb) solutions (i.e. Hemopure (OPK Biotech, Cambridge, MA), a glutaraldehyde polymerized bovine Hb; and PolyHeme (Northfield Laboratories Inc., Evanston, IL), a glutaraldehyde polymerized pyridoxylated human Hb) hamper further development. Both of these commercial products elicit vasoconstriction at the microcirculatory level, and lead to the development of systemic hypertension and oxidative tissue damage. These side-effects are hypothesized to occur either by a nitric oxide (NO) scavenging or oxygen (O2) oversupply mechanism, and are both exacerbated by PolyHb extravasation into the tissue space. In light of these 2 potential mechanisms, it is apparent that PolyHb size will have a profound impact on the extent of vasoconstriction, systemic hypertension and oxidative tissue toxicity. However, commercial PolyHb products are complex mixtures with broad size distributions defined only by the size cutoff of the ultrafiltration membranes used in their manufacture. Furthermore, these mixtures are known to contain up to 1% of individual tetrameric Hb molecules and a significantly higher proportion of lower molecular weight (MW) Hb oligomers (80% with MW < 500 kDa). Hence, the side-effects observed during clinical/pre-clinical trials are attributed to a mixture of low MW Hb polymers with different sizes and points of chemical modification, and not to any one, single PolyHb molecule. This precludes precise characterization of how individual components of these complex PolyHb mixtures interact with the vasculature. An important advance would therefore be the ability to produce molecularly uniform, monodisperse, and high MW PolyHb nanostructures. In this application, we hypothesize that the molecular diameter and topology of recombinant PolyHb (rPolyHb) will regulate vasoactivity and oxidative injury to tissues. To test our hypothesis we propose the following specific aims: Specific Aim 1: Use orthogonal split splicing inteins to produce well-defined, monodisperse, high MW rPolyHb nanostructures. Specific Aim 2a: Analyze the role of endothelial function on the development of vasoactivity and oxidative tissue injury to rPolyHbs of varying size. Specific Aim 2b: Evaluate the pharmacokinetics of rPolyHbs in normal guinea pigs and HFSD guinea pigs. Specific Aim 3: Evaluate the ability of rPolyHbs to restore tissue oxygenation and optimize survival in severe blood loss. The proposed work is both significant and innovative, since it seeks to develop safe and efficacious rPolyHbs for use in transfusion medicine. In addition, state-of-the-art biophysical techniques and two unique animal models will be used to understand rPolyHb physiological responses and determine the clinical potential of these novel materials.