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目的：构建刺槐素（Acacetin）的外泌体（Exosomes,EXOs）递药系统。方法：以人脐带间充质干细胞（human umbilical cord mesenchymal stem cells,hUMSCs）来源外泌体为药物载体，采用共孵育法、超声法和反复冻融法3种方式包载刺槐素，制备负载刺槐素的外泌体（EX-Aca）。采用高效液相色谱法（High Performance Liquid Chromatography,HPLC）测定EX-Aca包封率和载药率。透射电镜（TEM）、粒径分析和Western Blot对EXOs和EX-Aca的表征进行检测，通过体外毒性实验检测Ex-Aca的安全性，同时考察其细胞摄取能力。结果：通过高效液相色谱计算得出共孵育法包封率及载药率均高于超声法和反复冻融法。当刺槐素浓度为80μg/mL时，包封率和载药量最高，分别为91.90%和19.68%。透射电镜（TEM）下EXOs和EX-Aca均呈杯碟状，平均粒径分别为（102.356 96±0.656 23）nm和（117.300 38±0.747 83）nm，呈现典型外泌体特性。Western Blot均检测到EXOs和EX-Aca表面标志物CD9、ALIX和TSG101的蛋白表达。因此，EX-Aca的最优制备方法参数为：刺槐素浓度为80μg/mL，与hUMSCs来源外泌体4℃共孵育48 h。体外实验验证了EX-Aca对细胞低毒性，细胞存活率>94%且EX-Aca可被BV2细胞摄取。结论：成功构建刺槐素的外泌体递药系统，制备的EX-Aca对细胞具有低毒性，具有一定靶向性。
Abstract：Objective: To construct exosomes(EXOs) drug delivery system of acacetin.Methods: Using exosomes derived from human umbilical cord mesenchymal stem cells(h UMSCs) as drug carriers, The exosomes loaded with acacetin(EX-Aca)were prepared by co-incubation, ultrasound and repeated freeze-thaw methods. The encapsulation rate and loading rate of EX-Aca were determined by high performance liquid chromatography. Transmission electron microscopy(TEM), particle size analysis and Western Blot were used to detect the characterization of EXOs and EX-Aca. The safety of Ex-Aca was tested by in vitro toxicity test, and its cellular uptake capacity was also investigated. Result: The results of high performance liquid chromatography showed that the encapsulation rate and drug loading rate of co-incubation method were higher than that of ultrasonic method and repeated freeze-thaw method. When the concentration of acacetin was 80 μg/m L, the encapsulation rate and drug loading were the highest, which were 91.90% and 19.68%, respectively. Under transmission electron microscopy(TEM), EXOs and EX-Aca were cup-and-saucer-shaped, with average particle sizes of(102.356 96±0.656 23)nm and(117.300 38±0.747 83)nm, respectively, showing typical exosome characteristics. Western Blot detection of EXOs and EX-Aca surface markers CD9, ALIX and TSG101 protein expression. Therefore, the optimal preparation parameters of EX-Aca were as follows: The concentration of acacetin was 80 μg/m L, and h UMSCs-derived exosomes were incubated at 4 ℃ for 48 h. In vitro experiments confirmed that EX-Aca had low cytotoxicity, cell survival rate was > 94%, and EX-Aca could be taken up by BV2 cells.Conclusion: The exosomes drug delivery system of acacetin was successfully constructed, and the prepared EX-Aca had low toxicity to cells and certain targeting.
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基本信息:
DOI：10.13210/j.cnki.jhmu.20241101.002
中图分类号:R283.6
引用信息:
[1]张胜男,周玲,叶勒丹·马汉,等.人脐带间充质干细胞源外泌体负载刺槐素的制备方法研究[J].海南医科大学学报,2025,31(04):241-247+253.DOI:10.13210/j.cnki.jhmu.20241101.002.
基金信息:
新疆维吾尔自治区人民医院干细胞生物治疗基础和临床研究专项（GXBZX-2023007）; 新疆维吾尔自治区自然科学基金（2020D01C095）~~
2024-11-04
2024-11-04
2024-11-04