外泌体装载连翘酯苷A递药系统的制备及体外评价

Preparation and in vitro evaluation of forsythoside A-loaded exosomes

制备一种高生物相容性和低免疫原性的装载连翘酯苷A的外泌体(FTA-Exos)递药系统,并考察其对人肺上皮腺癌细胞A549细胞迁移能力的影响。该文采用超高速离心法结合超滤法提取纯化A549细胞中的外泌体,超声孵育法制备FTA-Exos,并通过马尔文粒度分析仪、透射电子显微镜和Western blot对FTA-Exos进行表征。激光共聚焦显微镜观察A549细胞对FTA-Exos的摄取,细胞划痕实验考察FTA-Exos对A549细胞水平迁移能力的影响。结果显示制备的FTA-Exos递药体系的平均粒径为(138.90±2.37) nm, PDI为0.291±0.013,平均电位为(-10.1±0.66) mV,FTA-Exos在载药后粒径略有增大。透射电子显微镜观察到FTA-Exos的外观形态上呈类球状,有明显的茶托样双层膜结构。Western blot结果显示,特异性蛋白CD63及Alix在外泌体中均有表达。激光共聚焦结果表明FTA-Exos能够被A549细胞摄取,在4 h时荧光显著增强,且能在细胞内稳定维持4~8 h。划痕实验显示其抑制A549细胞的迁移能力较连翘酯苷A显著增强(P<0.05)。该文初步成功构建了FTA-Exos递药系统,并证明其稳定性良好,制备方法稳定可靠,且具有良好的体外抑制A549细胞迁移作用,为后续深入研究及应用提供重要依据。

A drug delivery system of forsythoside A-loaded exosomes(FTA-Exos) with high biocompatibility and low immunogenicity was established to investigate its impact on the migration of human lung epithelial adenocarcinoma A549 cells. The exosomes from A549 cells were extracted and purified by ultra-high speed centrifugation and ultrafiltration. FTA-Exos were prepared by ultrasonic incubation, and characterized by particle size analysis, transmission electron microscopy, and Western blot assay. The uptake of FTA-Exos by A549 cells was observed under the laser confocal microscope, and the impact of FTA-Exos on the migration of A549 cells was investigated by cell scratch assay. The results showed that the average particle size of the prepared FTA-Exos was(138.90±2.37) nm, which increased slightly after drug loading. The PDI was 0.291±0.013, and the average potential was(-10.1±0.66) mV. The FTA-Exos were spheroidal in appearance as observed by transmission electron microscope, with an obvious saucer-like double-layer membrane. Western blot assay indicated that the specific proteins CD63 and Alix were both expressed in exosomes. The laser confocal microscopy suggested that FTA-Exos were taken up by A549 cells and stably maintained in the cell for 4-8 h, and the fluorescence was significantly enhanced at 4 h. The scratch assay showed that the inhibitory effect of FTA-Exos on the migration of A549 cells was significantly stronger than that of forsythoside A(P < 0.05). In conclusion, the drug delivery system of FTA-Exos established in this study had good stability, reliable preparation process, and potent inhibitory effect on the migration of A549 cells in vitro, which can provide an important reference for subsequent in-depth research and application.