Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Biomacromolecules. 2012 Sep 10;13(9):2748-59. doi: 10.1021/bm3007242. Epub 2012 Aug 17.
We synthesized extremely deformable red blood cell-like microgel particles and loaded them with bovine hemoglobin (Hb) to potentiate oxygen transport. With similar shape and size as red blood cells (RBCs), the particles were fabricated using the PRINT (particle replication in nonwetting templates) technique. Low cross-linking of the hydrogel resulted in very low mesh density for these particles, allowing passive diffusion of hemoglobin throughout the particles. Hb was secured in the particles through covalent conjugation of the lysine groups of Hb to carboxyl groups in the particles via EDC/NHS coupling. Confocal microscopy of particles bound to fluorescent dye-labeled Hb confirmed the uniform distribution of Hb throughout the particle interior, as opposed to the surface conjugation only. High loading ratios, up to 5 times the amount of Hb to polymer by weight, were obtained without a significant effect on particle stability and shape, though particle diameter decreased slightly with Hb conjugation. Analysis of the protein by circular dichroism (CD) spectroscopy showed that the secondary structure of Hb was unperturbed by conjugation to the particles. Methemoglobin in the particles could be maintained at a low level and the loaded Hb could still bind oxygen, as studied by UV-vis spectroscopy. Hb-loaded particles with moderate loading ratios demonstrated excellent deformability in microfluidic devices, easily deforming to pass through restricted pores half as wide as the diameter of the particles. The suspension of concentrated particles with a Hb concentration of 5.2 g/dL showed comparable viscosity to that of mouse blood, and the particles remained intact even after being sheared at a constant high rate (1000 1/s) for 10 min. Armed with the ability to control size, shape, deformability, and loading of Hb into RBC mimics, we will discuss the implications for artificial blood.
我们合成了具有高度变形能力的类似红细胞的微凝胶颗粒，并将其装载牛血红蛋白（Hb）以增强氧气传输。这些颗粒的形状和大小与红细胞（RBC）相似，是使用 PRINT（非润湿模板中的颗粒复制）技术制备的。水凝胶的低交联导致这些颗粒的网格密度非常低，允许血红蛋白通过被动扩散穿过颗粒。通过 EDC/NHS 偶联将血红蛋白的赖氨酸基团共价结合到颗粒中的羧基上，将血红蛋白固定在颗粒中。与荧光染料标记的血红蛋白结合的颗粒的共焦显微镜证实了血红蛋白在颗粒内部的均匀分布，而不是仅在表面结合。通过重量比高达 5 倍的血红蛋白与聚合物的高装载比，获得了对颗粒稳定性和形状没有显著影响的高装载比，尽管血红蛋白结合后颗粒直径略有减小。通过圆二色性（CD）光谱分析蛋白质，发现血红蛋白与颗粒结合后其二级结构未受干扰。通过紫外可见光谱研究发现，颗粒中的高铁血红蛋白可以保持在低水平，并且负载的血红蛋白仍然可以结合氧气。具有中等装载比的血红蛋白负载颗粒在微流控装置中表现出优异的变形能力，很容易变形通过比颗粒直径小一半的限制孔。血红蛋白浓度为 5.2 g/dL 的浓缩颗粒悬浮液的粘度与小鼠血液相当，即使在以恒定高剪切率（1000 1/s）剪切 10 分钟后，颗粒仍保持完整。由于能够控制血红蛋白进入 RBC 模拟物的大小、形状、变形能力和装载量，我们将讨论其对人造血的影响。
