SP94和CD47双修饰外泌体靶向递送PLK1siRNA治疗肝癌

Targeted delivery of PLK1 siRNA by SP94 and CD47 double-modified exosomes for liver cancer

目的设计一种基于外泌体高效递送的方法,将治疗性核酸递送到肝癌组织治疗原发性肝癌。方法构建靶向肝癌的融合表达SP94和CD47的质粒,转染至HEK293T细胞后提取分离外泌体。通过透射电子显微镜及纳米颗粒跟踪分析、蛋白质印迹法等方法对外泌体进行鉴定。小鼠经尾静脉分别注射200μg DiD标记的未修饰外泌体和SP94修饰的外泌体,采用小动物活体成像仪检测并分析外泌体对肝癌组织的靶向作用。将未修饰外泌体和CD47修饰的外泌体分别与巨噬细胞RAW264.7、小鼠肝癌细胞Hepa1-6共孵育,验证细胞对修饰后外泌体的吞噬情况。通过电穿孔法将Polo样激酶1(PLK1)siRNA加载到不同修饰的外泌体中,再与Hepa1-6细胞共孵育,采用流式细胞术检测细胞凋亡水平。原发性肝癌模型小鼠经尾静脉注射加载PLK1 siRNA的修饰外泌体,系统研究并分析其对原发性肝癌的治疗效果。结果SP94修饰增强了外泌体对肝癌组织的靶向作用,CD47修饰减少了外泌体被巨噬细胞吞噬。SP94和CD47双修饰外泌体递送PLK1 siRNA促进了肝癌细胞凋亡。在原发性肝癌模型小鼠中,SP94和CD47双修饰外泌体加载PLK1 siRNA能有效抑制小鼠肝癌结节的生长,延长小鼠生存期。结论SP94和CD47双修饰外泌体可以高效靶向肝癌递送治疗性核酸PLK1siRNA,有效抑制肝癌的生长。

Objective To develop an efficient exosome-based delivery method to effectively transport therapeutic nucleic acid to liver cancer tissue for the treatment of primary hepatic cancer.Methods HEK293T cells were transfected with a recombinant plasmid expressing SP94 and CD47,and the exosomes were extracted and isolated.Exosomes were identified by transmission electron microscope,nanometer particle tracking analysis,and Western blotting.The mice were injected with 200μg DiD labeled unmodified exosomes and SP94 modified exosomes via tail vein,and the targeting effect of exosomes on liver cancer tissue was detected and analyzed using small animal in vivo imaging.The unmodified exosomes and CD47 modified exosomes were incubated with macrophages(RAW264.7)and mouse hepatoma cells(Hepa1-6)to verify the phagocytosis of the modified exosomes by the cells.Polo-like kinase 1(PLK1)small interfering RNA(siRNA)was loaded into different modified exosomes by electroporation and then the exosomes were co-incubated with Hepal-6 cells,and the cell apoptosis was detected by flow cytometry.The modified exosomes loaded with PLK1 siRNA were injected into the tail vein to systematically study and analyze the therapeutic effect of PLK1 siRNA on primary hepatic cancer in mice.Results SP94 modification enhanced the targeting effect of exosomes on liver cancer tissue,and CD47 modification reduced the phagocytosis of exosomes by macrophages.PLK1 siRNA delivered by SP94 and CD47 double-modified exosomes increased the apoptosis of hepatoma cells.In the primary hepatic cancer mice,SP94 and CD47 double-modified exosomes loaded with PLK1 siRNA could effectively inhibit the growth of liver cancer nodules and prolong the survival time of tumorbearing mice.Conclusion SP94 and CD47 double-modified exosomes can efficiently deliver therapeutic nucleic acid PLK1 siRNA to target liver cancer and effectively inhibit the growth of liver cancer.