藤黄酸和雷帕霉素共载脂质体的制备及协同抗肿瘤活性研究

Preparation of gambogic acid and rapamycin co-loaded liposomes and study on the synergistic antitumor effect

目的制备共载藤黄酸和雷帕霉素的脂质体(liposomes co-loaded with gambogic acid and rapamycin,GR@Lip)并优化其处方,研究GR@Lip的体外抗肿瘤机制、体内药动学和生物分布。方法以包封率、载药量及粒径为评价指标,通过单因素和正交设计实验筛选GR@Lip的最佳处方,并对其进行表征和稳定性研究;通过CCK-8法和流式细胞术考察GR@Lip对肿瘤细胞增殖和凋亡的影响,细胞划痕实验与Transwell实验考察GR@Lip对肿瘤细胞迁移与侵袭的影响,透射电子显微镜(transmission electron microscope,TEM)和免疫荧光考察GR@Lip对肿瘤细胞自噬的影响,液相色谱-质谱联用技术(liquid chromotography with mass spectrometry,LC-MS)和活体成像仪研究GR@Lip的体内药动学和生物分布。结果GR@Lip的最佳处方为制备温度40℃,药脂比1∶1∶20,磷脂与胆固醇比例4∶1,超声功率195 W,超声时间5 min,水化介质为超纯水,水相pH值为7.1;该方法制备的GR@Lip藤黄酸包封率为(97.27±2.76)%,雷帕霉素包封率为(96.58±3.82)%,藤黄酸载药量为(3.29±0.44)%,雷帕霉素载药量为(4.91±0.44)%。TEM形态观察显示GR@Lip呈球形,动态光散射(dynamic light scattering,DLS)检测其平均粒径为(157.19±1.74)nm、ζ电位为(−22.1±1.3)mV,且具有良好的稳定性。体外抗肿瘤活性实验结果显示,藤黄酸和雷帕霉素联用能协同抑制肿瘤细胞增殖、迁移和侵袭,并显著促进肿瘤细胞凋亡,增强自噬。此外,体内药动学和生物分布结果显示,GR@Lip可在体内滞留更长时间且具有良好的肿瘤靶向性。结论成功制备了GR@Lip,揭示其具有通过多途径协同抗肿瘤的活性,并显著改善了藤黄酸和雷帕霉素的药动学行为,为进一步体内研究和未来临床应用提供了实验依据。

Objective To prepare liposomes co-loaded with gambogic acid and rapamycin(GR@Lip),optimize its prescription,perform anti-tumor mechanism research in vitro,pharmacokinetics and biodistribution in vivo.Methods The entrapment efficiency,drug loading,particle size were used as evaluation indicators.Single factor investigation method and orthogonal design experiments were used to optimize the optimum formulation of GR@Lip,and its characterization and stability were studied.The effects of GR@Lip on the proliferation and apoptosis of tumor cells were investigated by CCK-8 and flow cytometry.The effects of GR@Lip on the migration and invasion of tumour cells were investigated by cell scratching,Transwell.The effects of GR@Lip on the autophagy of tumour cells were investigated by transmission electron microscope(TEM)and immunofluorescence.The effects of GR@Lip on the pharmacokinetics and biodistribution in vivo were investigated by liquid chromotography with mass spectrometry(LC-MS)and living body imager.Results The optimal conditions were determined as follows:temperature is 40℃,ratio of drug to lipid is 1:1:20,the phospholipid-cholesterol ratio is 4:1,ultrasonic power is 195 W,ultrasonic time is 5 min,hydration medium is ultrapure water and aqueous pH value is 7.1.The entrapment efficiency and drug loading of gambogic acid and rapamycin were(97.27±2.76)%,(96.58±3.82)%,(3.29±0.44)%and(4.91±0.44)%,respectively.GR@Lip showed a spherical under TEM with an average particle size of(157.19±1.74)nm,ζpotential was(−22.1±1.3)mV by dynamic light scattering(DLS),and had good stability.In vitro antitumor activity experiment showed that gambogic acid combination rapamycin can inhibit the proliferation,migration and invasion of tumor cells,and also significantly promote apoptosis and autophagy.In addition,pharmacokinetics and biodistribution results in vivo showed that GR@Lip can remain for long time and had good tumor targeting.Conclusion This study successfully prepare GR@Lip,revealing that it has synergistic anti-tumor activity through multiple pathways,and significantly improving the pharmacokinetic behavior of gambogic acid and rapamycin in vivo,providing a basis for further in vivo research and future clinical applications.