高效携氧人工红细胞的构建及其机制的研究

The ever-increasing demands for blood in clinics and dilemma faced by conventional blood transfusion have attracted more and more interests from international scientists, especially, the chemists, on the study of artificial red blood cells (ARBCs), which has become a hot research field. In this climate, the construction of ARBC with high-efficient oxygen carrying capacity is very meaningful for satisfaction of patients with postoperative hypoxic-ischemia and cardiorespiratory failure as well as physiological requirements of special people in aeronautics & astronautics as well as deep sea diving. Based on our previous investigation on layer-by-layer assembled hemoglobin {Hb/NPs}n and direct electrochemistry of red blood cells, through combination of electrochemical analysis, cell biology, nanotechnology and safety toxicity, this proposal will apply techniques of self-assembly and microcapsule to construct aggregates of ordered hemoglobin ({Hb/NPs}n) and then encapsulate them in biocompatible microcapsules to form ARBCs, which are similar to natural red blood cells. The obtained ARBC will be assembled at the surface of working electrodes for the study of their direct electrochemistry and harvest of thermodynamic and dynamic data. ARBC with high-efficiency oxygen carrying capacity would be fabricated and its mechanism would be elucidated by coupling with physiological and biochemical analysis and optimizing experimental parameters. Meanwhile, evaluation of biosafety and toxicity of ARBCs would be performed through in vivo and in vitro examinations in order to achieve ARBCs with better biocompatibility and strong oxygen carrying capacity.

临床医疗对血液需求日益增加，且传统输血方式面临种种困境，这使得人工红细胞越来越受到国内外学者，尤其是化学工作者广泛关注，成为研究热点。而构筑高效携氧能力的人工红细胞，满足术后缺血缺氧、心肺功能衰竭等乃至航空航天、深海潜水等特殊人群的生理需求则更有意义。 在对血红蛋白层层有序组装{Hb/NPs}n及红细胞直接电化学前期研究基础上，结合电分析化学、细胞生物学、纳米和安全性毒理学评价等技术，本项目用自组装和微囊包裹等方法，构筑有序血红蛋白聚集体{Hb/NPs}n，并将其包裹到生物兼容性好的微囊中，形成类似于天然红细胞，含有大量{Hb/NPs}n的人工红细胞。将其组装在电极表面，研究其直接电化学，获取热力学和动力学数据，结合生理生化分析，优化实验，构筑高效携氧能力的人工红细胞，并阐述其携氧增强机制；同时通过动物体内、体外试验进行安全性毒理学评价，最终获得生物兼容性好、携氧能力强的人工红细胞。

传统输血方式面临种种困境，临床医疗对血液需求日益增加，高效携氧的人工红细胞（ARBCs）成为研究热点。.一方面，将血红蛋白（Hb）与纳米材料（NPs）层层组装，构建有序的{Hb/NPs}n聚集体，如：{Hb/Au}3，{Hb/CS@Fe3O4}n，{Hb/CG}n。运用电化学方法及紫外光谱法研究有序{Hb/NPs}n集聚体的携氧能力，阐述携氧增强机制，发现集聚体的携氧能力与组装层数有关，在磁场的诱导下，可实现有序排列，使得标准熵变小，有利于Hb与更多的氧气结合，从而增强携氧能力。.另一方面，以微纳米结构的模板颗粒（碳酸钙、碳酸锰、氢氧化钙）为载体，通过Hb与葡聚糖、壳聚糖、海藻酸钠、聚多巴胺等成膜材料和模板之间的有机结合，利用层层自组装、微囊包裹法、双重乳液法、直接构筑法等方法，构筑了不同形状（球形、椭球形、双凹碟形）、不同大小（0.9-6 m）的ARBCs。运用电化学技术、紫外-可见光谱技术等研究ARBCs的“携氧-释氧”动力学过程及携氧机理，发现ARBCs能可逆地“结合和释放”氧气，稳定性良好，且37℃时的携氧和释氧量最高，而添加抗氧化酶的ARBCs可为细胞提供充足的有氧环境，抑制自由基产生，解决局部乏氧和氧化应激损伤。因此，以电化学为主要手段，辅以光学、谱学、生物学等技术，建立了系统评价ARBCs载氧功能的新方法。.此外，将制备的集聚体和ARBCs组装到电极表面，研究其电化学行为，获取电子转移速率、熵变等热力学和动力学数据，发现其标准熵比天然Hb的标准熵要小得多，表明ARBCs的热稳定性较好，其内部Hb排列有序。.最后，通过体外的细胞试验和体内的动物试验，评价了ARBCs的毒理学及生物安全性，发现其安全无毒，对试验动物的血液指标无影响，对心、肝、肾、肺等脏器均无损伤，为临床应用奠定了基础，可望满足术后缺血缺氧、心肺功能衰竭等乃至航空航天、深海潜水等特殊人群的生理需求。