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  

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

作　　者：杨康 赵会民[2] 樊毫军 苏谐 伍国俊 Yang Kang;Zhao Huimin;Fan Haojun;Su Xie;Wu Guojun(Department of Emergency,Second Affiliated Hospital of Guangxi Medical University,Nanning 530007,Guangxi Zhuang Autonomous Region,China;Trauma Center,Second Affiliated Hospital of Guangxi Medical University,Nanning 530007,Guangxi Zhuang Autonomous Region,China;Institute of Disaster Medicine,Tianjin University,Tianjin University,Tianjin 300072,China;Department of Hematology,Second Affiliated Hospital of Guangxi Medical University,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]天津大学应急医学研究院,天津市300072 [4]广西医科大学第二附属医院血液内科,广西壮族自治区南宁市530007 [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

出　　处：《中国组织工程研究》2022年第21期3369-3374,共6页Chinese Journal of Tissue Engineering Research

基　　金：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。

摘　　要：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800-1000 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。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 cult

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 

分 类 号：R457.1[医药卫生—治疗学] R318[医药卫生—临床医学]