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  

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

作　　者：黄志华 赵会民[2] 苏春元 杨康 Huang Zhihua;Zhao Huimin;Su Chunyuan;Yang Kang(Department of Emergency,Second Affiliated Hospital of Guangxi Medical University,Nanning 530007,Guangxi Zhuang;Trauma Center,Second Affiliated Hospital of Guangxi Medical University,Nanning 530007,Guangxi Zhuang Autonomous Region,China;Guangxi Key Laboratory of Mediterranean Hemoglobinopathy Prevention and Treatment,Nanning 530007,Guangxi Zhuang Autonomous Region,China;Guangxi Key Emergency and Critical Care Medicine Laboratory,Nanning 530007,Guangxi Zhuang Autonomous Region,China)

机构地区：[1]广西医科大学第二附属医院急诊科,广西壮族自治区南宁市530007 [2]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 [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

出　　处：《中国组织工程研究》2025年第22期4740-4747,共8页Chinese Journal of Tissue Engineering Research

基　　金：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。

摘　　要：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

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 

分 类 号：R459.9[医药卫生—治疗学] R319[医药卫生—临床医学] R826.26