中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (22): 4740-4747.doi: 10.12307/2025.457

• 纳米生物材料 nanobiomaterials • 上一篇    下一篇

一种多重修饰血紫蛋白纳米氧载体的构建及体外性能检测

黄志华1 ，赵会民2 ，苏春元3 ，杨  康4   

  1. 广西医科大学第二附属医院，1急诊科，2创伤中心，广西壮族自治区南宁市   530007；3广西地中海贫血防治重点实验室，广西壮族自治区南宁市   530007；4广西重点急危重症医学实验室，广西壮族自治区南宁市   530007
  • 收稿日期:2024-03-26 接受日期:2024-06-01 出版日期:2025-08-08 发布日期:2024-12-06
  • 通讯作者: 赵会民，博士，主任医师，广西医科大学第二附属医院创伤中心，广西壮族自治区南宁市 530007
  • 作者简介:黄志华，男，1996年生，湖南省邵阳市人，汉族，广西医科大学在读硕士，主要从事血红蛋白氧载体研究。
  • 基金资助:
    国家自然科学基金项目(82260047)，项目负责人：赵会民；广西自然科学基金项目 (2023GXNSFAA026168)，项目负责人：赵会民；广西壮族自治区重点临床专科(创伤外科)研发专项项目(GKTS202203101)，项目负责人：赵会民

Construction and in vitro performance testing of a multi-modified hemerythrin-based nano-oxygen carrier

Huang Zhihua1, Zhao Huimin2, Su Chunyuan3, Yang Kang4   

  1. 1Department of Emergency, 2Trauma Center, Second Affiliated Hospital of Guangxi Medical University, Nanning 530007, Guangxi Zhuang Autonomous Region, China; 3Guangxi Key Laboratory of Mediterranean Hemoglobinopathy Prevention and Treatment, Nanning 530007, Guangxi Zhuang Autonomous Region, China; 4Guangxi Key Emergency and Critical Care Medicine Laboratory, Nanning 530007, Guangxi Zhuang Autonomous Region, China
  • Received:2024-03-26 Accepted:2024-06-01 Online:2025-08-08 Published:2024-12-06
  • Contact: Zhao Huimin, MD, Chief physician, Trauma Center, Second Affiliated Hospital of Guangxi Medical University, Nanning 530007, Guangxi Zhuang Autonomous Region, China
  • About author:Huang Zhihua, Master candidate, Department of Emergency, Second Affiliated Hospital of Guangxi Medical University, Nanning 530007, Guangxi Zhuang Autonomous Region, China
  • Supported by:
    National Natural Science Foundation of China, No. 82260047 (to ZHM); Guangxi Natural Science Foundation, No. 2023GXNSFAA026168 (to ZHM); Guangxi Zhuang Autonomous Region Key Clinical Specialty (Trauma Surgery) Research & Development Special Project, No. GKTS202203101 (to ZHM)

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摘要：


文题释义：

红细胞替代物：是一种能满足临床输血、离体器官维护、灾难救援、战场救护等用血需求的功能用品，主要包括以全氟化碳为代表的高溶氧液体和基于血红蛋白分子化学修饰或合成膜封装的氧载体。与红细胞输注相比，红细胞替代物能减少病毒的传播风险，使用前无需进行交叉配血，并且保质期较长。
血红蛋白氧载体：多以猪、牛等哺乳动物血红蛋白为基础，通过聚合、加成、偶联、共轭等化学修饰或通过脂质体、高分子聚合物、金属骨架有机物、多孔二氧化硅等人工合成膜纳米封装技术去除游离血红蛋白的毒副作用，成为安全高效的红细胞替代用品。


背景：血紫蛋白分子稳定性及生物相容性优于人和哺乳动物血红蛋白，经过修饰可能成为更加安全长效的红细胞代用品。

目的：制备多重修饰血紫蛋白纳米微粒，进行理化性质表征及体外性能测试。
方法：以切向流超滤法分离纯化方格星虫血紫蛋白，使用京尼平完成分子内交联，然后利用多巴胺完成纳米粒子封装，再用聚乙二醇完成钝化，获得多重修饰血紫蛋白纳米粒，表征该纳米粒的理化性质。将不同质量浓度(0，0.25，0.5，1.0，2.0 mg/mL)的血紫蛋白纳米粒、血紫蛋白、血红蛋白氧载体HBOC-201分别与巨噬细胞共同孵育6，24 h，与血管内皮细胞共同孵育24 h，采用CCK-8法检测细胞存活率，ELISA法检测血管内皮细胞培养液中一氧化氮及血管细胞黏附因子1水平。

结果与结论：①血紫蛋白纳米粒电镜下呈现椭球形，有致密外膜，内部质地较为均匀，粒径为(150.12±1.67) nm，分散指数为0.21±0.03，Zeta电位为(-24.54±2.61) mV，半饱和氧分压为(0.97±0.15) kPa，Hill系数为1.49±0.16。②孵育6 h，在≤1.0 mg/mL质量浓度范围内，血紫蛋白纳米粒组、血紫蛋白组、HBOC-201组巨噬细胞存活率均在85%以上；在2.0 mg/mL质量浓度下，仅血紫蛋白纳米粒组巨噬细胞存活率在80%以上。孵育24 h，3组巨噬细胞存活率均低于80%，其中血紫蛋白纳米粒组巨噬细胞存活率高于血紫蛋白组、HBOC-201组(P < 0.05)。③随着药物质量浓度的增加，3组血管内皮细胞存活率降低，在1.0 mg/mL或2.0 mg/mL质量浓度下，血紫蛋白纳米粒组细胞存活率高于血紫蛋白组、HBOC-201组(P < 0.05)；在相同质量浓度下，血紫蛋白纳米粒组一氧化氮水平高于血紫蛋白组、HBOC-201组(P < 0.05)；在0.25-2.0 mg/mL质量浓度范围内，血紫蛋白纳米粒组血管细胞黏附因子1水平低于血紫蛋白组、HBOC-201组(P < 0.05)。④结果表明，经过分子内交联和聚多巴胺/聚乙二醇修饰的血紫蛋白纳米粒在体外具有良好的携氧活性，抗吞噬性能更优，细胞毒性更小。

https://orcid.org/0009-0004-9409-8201 (黄志华)

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料；口腔生物材料；纳米材料；缓释材料；材料相容性；组织工程

关键词: 红细胞代用品, 血紫蛋白, 化学修饰, 细胞实验, 血红蛋白氧载体

Abstract: BACKGROUND: Molecular stability and biocompatibility of hemerythrin surpass those of human and mammalian hemoglobin, making it a potential candidate for a safer and more effective erythrocyte substitute after modification.
OBJECTIVE: To prepare multi-modified hemerythrin nanoparticles, characterize them, and test their performance in vitro. 
METHODS: The hemerythrin of Sipunculus sphenodontus was separated and purified by tangential flow ultrafiltration. The intramolecular cross-linking was completed by genipin. The nanoparticles were encapsulated by dopamine, and passivated by polyethylene glycol to obtain multi-modified hemerythrin nanoparticles. The physicochemical properties of the nanoparticles were characterized. Hemerythrin nanoparticles, hemerythrin, and hemoglobin oxygen carrier HBOC-201 with different mass concentrations (0, 0.25, 0.5, 1.0, and 2.0 mg/mL) were incubated with macrophages for 6 and 24 hours, and with endothelial cells for 24 hours. The cell survival rate was detected by CCK-8 assay. The levels of nitric oxide and vascular cell adhesion factor 1 in the culture medium of endothelial cells were detected by ELISA.
RESULTS AND CONCLUSION: (1) Under electron microscopy, hemerythrin nanoparticles were ellipsoidal, with a dense outer membrane and a relatively uniform internal texture. The particle size was (150.12±1.67) nm; the dispersion index was 0.21±0.03; the Zeta potential was (-24.54±2.61) mV; the half-saturated oxygen partial pressure was (0.97±0.15) kPa, and the Hill coefficient was 1.49±0.16. (2) After incubation for 6 hours, within the mass concentration range of ≤1.0 mg/mL, the survival rates of macrophages in the hemerythrin nanoparticle group, the hemerythrin group, and the HBOC-201 group were all above 85%. At a mass concentration of 2.0 mg/mL, only the survival rate of macrophages in the hemerythrin nanoparticle group was above 80%. After incubation for 24 hours, the survival rates of macrophages in the three groups were all lower than 80%, among which the survival rate of macrophages in the hemerythrin nanoparticle group was higher than that in the hemerythrin group and the HBOC-201 group (P < 0.05). (3) With the increase of drug concentration, the survival rate of vascular endothelial cells in the three groups decreased. At 1.0 mg/mL or 2.0 mg/mL mass concentration, the survival rate of cells in the hemerythrin nanoparticle group was higher than that in the hemerythrin group and HBOC-201 group (P < 0.05). At the same mass concentration, the nitric oxide level in the hemerythrin nanoparticle group was higher than that in the hemerythrin group and HBOC-201 group (P < 0.05). In the range of 0.25-2.0 mg/mL mass concentration, the vascular cell adhesion factor 1 level in the hemerythrin nanoparticle group was lower than that in the hemerythrin group and HBOC-201 group (P < 0.05). (4) The results showed that the hemerythrin nanoparticles modified with intramolecular cross-linking and polydopamine/polyethylene glycol had good oxygen-carrying activity in vitro, better anti-phagocytic performance, and less cytotoxicity.

Key words: erythrocyte substitutes, hemerythrin, chemical modification, cell experiments, hemoglobin-based oxygen carriers

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