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水蛭素-草酸二维生素E酯脂质体的构建及其体外抗凝作用评价
Construction of hydrogen peroxide-scavenging liposomes based on vitamin E and evaluation of its
in vitro anticoagulant effect- 陆军军医大学学报 2024年46卷第21期 页码：2475-2484
- 作者机构：
1.400038 重庆，陆军军医大学(第三军医大学)第一附属医院神经内科
2.400038 重庆，陆军军医大学(第三军医大学)药学与检验医学系药剂学教研室
- 作者简介：
[周振华，E-mail：zhouzhenhua@tmmu.edu.cn ZHOU Zhenhua, E-mail: zhouzhenhua@tmmu.edu.cn
- 基金信息：国家自然科学基金青年基金项目(82301516)
DOI：10.16016/j.2097-0927.202404044
中图分类号： R914.5;R967;R977.25收稿：2024-04-12，
修回：2024-05-13，
纸质出版：2024-11-15
- 稿件说明：
移动端阅览
杨清华, 张建祥, 周振华. 水蛭素-草酸二维生素E酯脂质体的构建及其体外抗凝作用评价[J]. 陆军军医大学学报, 2024,46(21):2475-2484.
Qinghua YANG, Jianxiang ZHANG, Zhenhua ZHOU. Construction of hydrogen peroxide-scavenging liposomes based on vitamin E and evaluation of its
in vitro anticoagulant effect[J]. Journal of Army Medical University, 2024, 46(21): 2475-2484.杨清华, 张建祥, 周振华. 水蛭素-草酸二维生素E酯脂质体的构建及其体外抗凝作用评价[J]. 陆军军医大学学报, 2024,46(21):2475-2484. DOI： 10.16016/j.2097-0927.202404044.
Qinghua YANG, Jianxiang ZHANG, Zhenhua ZHOU. Construction of hydrogen peroxide-scavenging liposomes based on vitamin E and evaluation of its
in vitro anticoagulant effect[J]. Journal of Army Medical University, 2024, 46(21): 2475-2484. DOI： 10.16016/j.2097-0927.202404044.
摘要
目的
2
基于维生素E，构建同时具有过氧化氢清除功能和抗凝作用的脂质体，探讨其是否可以作为急性缺血性脑卒中的治疗方式并评价其体外抗凝作用。
方法
2
利用过氧草酸酯键将两分子维生素E(生育酚，VE)化学键合，得到目标化合物草酸二维生素E酯(oxalate vitamin E，OVE)；通过酰胺化反应将抗凝药物水蛭素(hirudin)连接到亚油酸(linoleic acid
LA)得到抗凝目标单元亚油酸-水蛭素(LA-hirudin
LH)；并通过红外光谱、核磁等手段对所合成的产物进行结构表征。采用薄膜分散法制备水蛭素-草酸二维生素E酯脂质体(hirudin-OVE liposome
HOL)，采用激光粒度仪(DLS)和透射电子显微镜(TEM)等方法对脂质体进行表征。选取小鼠脑微血管内皮细胞(mouse brain-derived endothelial cells
bEnd3)和人脐静脉内皮细胞(human umbilical vein endothelial cells
HUVEC)进行细胞实验。通过ELISA测定过氧化氢以及细胞炎症因子TNF-α、IL-6和IL-1β水平，以评价脂质体对细胞氧化应激和炎症水平的影响。采用细胞计数实验(CCK-8)和细胞增殖检测实验(Edu)考察脂质体对细胞数目和细胞增殖情况的影响。取小鼠全血，考察所制备脂质体的抗凝作用。
结果
2
核磁结果表明成功合成了OVE，红外谱图结果表明水蛭素和亚油酸成功键合得到亚油酸-水蛭素。透射电镜和激光粒度仪结果表明所制备的脂质体形状为类球形，且粒径分布均匀，平均粒径为161 nm。细胞实验表明，脂质体能够显著抑制模型细胞分泌过氧化氢和炎症因子，具有明显的抗氧化、抗炎作用(
P
＜0.05)。细胞计数实验和细胞增殖检测实验结果证明脂质体能够抑制炎症导致的细胞数目减少、缓解LPS刺激所引起的细胞增殖抑制(
P
＜0.01)。抗凝实验结果表明，脂质体能够显著抑制CaCl
2
诱导的凝血反应(
P
＜0.01)。
结论
2
基于维生素E成功合成过氧化氢清除性材料OVE，并以抗凝药物水蛭素和OVE制备OVE抗凝脂质体(HOL)，该脂质体在细胞层面上具有良好的抗氧化、抗炎效果，并具有显著的抗凝作用。
Abstract
Objective
2
To construct vitamin E (tocopherol
VE)-based liposomes with both hydrogen peroxide-scavenging function and anticoagulant effect
and explore whether they can be used to treat acute ischemic stroke and then evaluate their
in vitro
anticoagulant effect.
Methods
2
Two molecules of VE were chemically bonded through peroxalate bond to obtain oxalate vitamin E (OVE). The anticoagulant unit LA-Hirudin was obtained by amidation reaction by connecting the an
ticoagulant drug hirudin to linoleic acid (LA). The structures of the synthesized products were characterized by using H nuclear magnetic resonance (
1
HNMR) and FT-IR spectra. Hirudin-Oxalic acid divitamin E ester liposome (HOL) was prepared by thin film dispersion and characterized with dynamic light scattering (DLS) and transmission electron microscopy (TEM). Mouse brain-derived endothelial cells (bEnd3) and human umbilical vein endothelial cells (HUVEC) were applied as the object of cell experiments. The levels of H
2
O
2
and cellular inflammatory factors TNF-α
IL-6 and IL-1β were determined by ELISA to evaluate the effect of the liposomes on the severity of cellular inflammation. Cell counting kit-8 (CCK-8) assay and cell proliferation assay (5-Ethynyl-2′-deoxyuridine
Edu) were used to detect the effects of the liposomes on cell count and proliferation. The anticoagulant effect of the prepared liposomes was detected by using whole blood of mice.
Results
2
1
HNMR spectrum showed that OVE was successfully synthesized
and the FT-IR spectrum displayed that hirudin and linoleic acid were successfully bonded to obtain LA-Hirudin. The results of DLS and TEM indicated that the prepared liposomes were spheroids
in uniform distribution
with an average particle size of 161 nm. The cell experiments showed that the liposomes significantly inhibited the secretion of H
2
O
2
and inflammatory factors
and exerted obvious antioxidant and anti-inflammatory effects in the model cells (
P
<
0.05). CCK-8 assay and Edu proliferation assay proved that the liposomes suppressed the decreased cell count and alleviated the inhibited cell proliferation caused by LPS (
P
<
0.01). Anticoagulation experiment revealed that the liposomes had significant inhibitory effect of CaCl
2
-induced coagulation reaction (
P
<
0.01).
Conclusion
2
A hydrogen peroxide-scavenging function material
OVE is synthesized
and then anticoagulant drug hirudin and OVE are used to prepare OVE thrombolytic liposomes
HOL. The liposomes have antioxidant and anti-inflammatory effects at the cellular level and also have significant anticoagulant effect.
关键词
Keywords
references
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