中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (21): 3369-3374.doi: 10.12307/2022.646

• 材料生物相容性 material biocompatibility • 上一篇    下一篇

聚多巴胺修饰蚯蚓血红蛋白纳米氧载体的构建及性能检测

杨  康1，赵会民2，樊毫军3，苏  谐4，伍国俊5   

  1. 广西医科大学第二附属医院，1急诊科，2创伤中心，4血液内科，广西壮族自治区南宁市  530007；3天津大学应急医学研究院，天津市  300072；5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:2021-07-28 接受日期:2021-10-23 出版日期:2022-07-28 发布日期:2022-01-28
  • 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 樊毫军，博士，教授，天津大学应急医学研究院，天津市 300072
  • 作者简介:杨康，男，1993年生，湖北省孝感市人，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:
    国家重点研发计划重点专项(2018YFC1504402-02)，项目负责人：樊毫军；广西医科大学第二附属医院基金项目(EFYKY2020010)，项目负责人：赵会民

Preparation of a polydopamine modified hemerythrin-based nano-oxygen carrier and performance testing

Yang Kang1, Zhao Huimin2, Fan Haojun3, Su Xie4, Wu Guojun5   

  1. 1Department of Emergency, 2Trauma Center, 4Department of Hematology, Second Affiliated Hospital of Guangxi Medical University, Nanning 530007, Guangxi Zhuang Autonomous Region, China; 3Institute of Disaster Medicine, Tianjin University, Tianjin 300072, China; 5Guangxi Key Emergency and Critical Care Medicine Laboratory, Nanning 530007, Guangxi Zhuang Autonomous Region, China 
  • Received:2021-07-28 Accepted:2021-10-23 Online:2022-07-28 Published:2022-01-28
  • Contact: Zhao Huimin, MD, Chief physician, Trauma Center, Second Affiliated Hospital of Guangxi Medical University, Nanning 530007, Guangxi Zhuang Autonomous Region, China Fan Haojun, MD, Professor, Institute of Disaster Medicine, Tianjin University, Tianjin 300072, China
  • About author:Yang Kang, Master candidate, Department of Emergency, Second Affiliated Hospital of Guangxi Medical University, Nanning 530007, Guangxi Zhuang Autonomous Region, China
  • Supported by:
    the National Key Research and Development Program of China, No. 2018YFC1504402-02 (to FHJ); the Fund Project of the Second Affiliated Hospital of Guangxi Medical University, No. EFYKY2020010 (to ZHM)

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摘要：

文题释义：
血红蛋白氧载体：以血红蛋白为原料，通过交联、聚合、偶联等化学修饰或者将其封装在人工合成的膜中，得到一种具有运载氧气能力的红细胞替代物。
红细胞替代物：通过模拟天然红细胞生物学功能的氧载体，主要包括基于全氟碳的乳剂、基于含铁(Fe2+)卟啉等血红蛋白的氧载体，与红细胞相比具有降低感染风险、延长储存时间、使用时无需配型等优点。
蚯蚓血红蛋白：存在于蚯蚓、星虫等低等生物体内的一类大分子呼吸蛋白，不依赖红细胞发挥生理性携氧作用。

背景：蚯蚓血红蛋白分子稳定性和血管毒性反应优于哺乳动物血红蛋白，聚多巴胺改性可能进一步发挥其作为血红蛋白氧载体的优势，目前国内外尚未报道。  
目的：制备聚多巴胺修饰蚯蚓血红蛋白纳米氧载体，并检测其性能。
方法：从赤子爱胜蚓分离提纯血红蛋白，以牛血红蛋白为对照，分别与盐酸多巴胺按1∶2质量比在pH=8.5-8.8条件下完成修饰改性，分别构建聚多巴胺表面修饰蚯蚓血红蛋白纳米微粒(PDA-HrNPs)与聚多巴胺表面修饰牛血红蛋白纳米微粒(PDA-BHbNPs)，检测PDA-HrNPs的纳米微粒直径、离散度、Z电位、功能基团结构、P50及Hill系数。将不同质量浓度[0(正常对照)，1.0，2.5，4.0，5.5 g/L]的两组纳米微粒分别与血管内皮细胞或巨噬细胞共培养，培养6，12，24 h后，检测巨噬细胞存活率；培养24 h后，检测血管内皮细胞存活率及培养液中NO浓度。
结果与结论：①蚯蚓血红蛋白经聚多巴胺修饰后，红外光谱显示酰胺键谱带Ⅰ与Ⅱ持续存在，在800-1 000 cm-1之间出现聚多巴胺与血红蛋白分子交联生成的新基团谱带，粒径(202.2±13.64)nm，分散指数为0.22±0.05，Z电位(1.28±0.87)mV，P50为(1.44±0.02)kPa，Hill系数1.58±0.02；②两种纳米微粒培养的巨噬细胞存活率均低于正常对照组(P < 0.05)，随着培养时间的延长，两种纳米微粒培养的巨噬细胞存活率下降；相同质量浓度下(2.5，4.0，5.5 g/L)，PDA-HrNPs组培养6 h后的巨噬细胞存活率高于PDA-BHbNPs组(P < 0.05)；③培养24 h后，随着纳米微粒质量浓度的增加，两种纳米微粒培养的血管内皮细胞的存活率与培养液NO浓度下降，相同质量浓度下两种纳米微粒培养细胞的存活率与培养液NO浓度比较差异均无显著性意义(P > 0.05)；④结果表明，聚多巴胺蚯蚓血红蛋白纳米微粒在体外具有良好的携氧活性与安全性，抗吞噬性能优于聚多巴胺修饰牛血红蛋白纳米微粒。
缩略语：聚多巴胺修饰蚯蚓血红蛋白纳米微粒：polydopamine modified hemerythrin nanoparticles，PDA-HrNPs；聚多巴胺修饰牛血红蛋白纳米微粒：polydopamine modified bovine hemoglobin nanoparticles，PDA-BHbNPs

https://orcid.org/0000-0003-4004-115X (杨康) 

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: 红细胞替代物, 血红蛋白载体, 蚯蚓血红蛋白, 化学修饰, 纳米微粒, 细胞学实验

Abstract: BACKGROUND: Hemerythrin may be superior to mammalian hemoglobin in terms of molecular stability and low toxicity. Polydopamine modification may further exert its advantages as a hemoglobin oxygen carrier; however, it has not been reported yet. 
OBJECTIVE: To prepare a polydopamine modified hemerythrin-based nano-oxygen carrier and examine its performance. 
METHODS: The sample of hemerythrin was purified from Eisenia foetida, and bovine hemoglobin was used as a control. The chemical modification reaction was completed with dopamine hydrochloride in a 1:2 mass ratio under pH=8.5-8.8 condition to construct polydopamine modified hemerythrin nanoparticles (PDA-HrNPs) and polydopamine modified bovine hemoglobin nanoparticles (PDA-BHbNPs). The diameter, polymer dispersity index, Z potential, functional molecular group, P50, and Hill coefficient were measured. Two groups of nanoparticles with different mass concentrations [0 (normal control), 1.0, 2.5, 4.0, 5.5 g/L] were co-cultured with vascular endothelial cells or macrophages, respectively. The survival rate of macrophages was measured at 6, 12, and 24 hours after culture. The survival rate of vascular endothelial cells and the concentration of NO in the culture medium were measured at 24 hours after culture. 
RESULTS AND CONCLUSION: (1) Infrared spectroscopy of PDA-HrNPs showed that the amide bond bands I and II continued to exist, and a new group band formed by the cross-linking of polydopamine and hemerythrin molecules appeared between 800 and 1000 cm-1. PDA-HrNPs particle size: (202.2±13.64) nm, polymer dispersity index: 0.22±0.05, Z potential: (1.28±0.87) mV, P50: (1.44±0.02) kPa, Hill coefficient: 1.58±0.02. (2) The survival rate of macrophages cultured with two kinds of nanoparticles was lower than that of the normal control group (P < 0.05). The survival rate of macrophages decreased with the increase in time. The survival rate of macrophages at 6 hours in the PDA-HrNPs group was significantly better than that in the PDA-BHbNPs group at the same concentration (2.5, 4.0, and 5.5 g/L) (P < 0.05). (3) After 24 hours of culture, with the increase in the mass concentration of nanoparticles, the survival rate of vascular endothelial cells cultured with two kinds of nanoparticles and the NO concentration in the culture medium decreased. There was no significant difference in the survival rate of cultured cells and the NO concentration in the culture medium between the two kinds of nanoparticles at the same mass concentration (P > 0.05). (4) It is shown that the PDA-HrNPs have good oxygen-carrying activity and safety. The antiphagocytic performance of PDA-HrNPs is better than that of PDA-BHbNPs. 

Key words: erythrocyte substitute, hemoglobin-based oxygen carrier, hemerythrin, chemical modification, nanoparticle, cytology test

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