双配体修饰的阿霉素脂质体靶向于脑胶质瘤的体外研究

Study of Doxorubicin Liposome Modified with Transferrin and Folic Acid Targeting to Glioma in Vitro

关键词:  阿霉素  转铁蛋白  叶酸  脂质体  胶质瘤

Key words:  doxorubincin  transferrin  folic acid  liposome  glioma

摘要:

目的 筛选和优化转铁蛋白、叶酸共同修饰的阿霉素脂质体的处方及制备工艺，以期得到具有良好的脑胶质瘤靶向治疗作用的给药系统。方法 采用薄膜分散和硫酸铵梯度法制备阿霉素脂质体。将叶酸连接至二硬脂酸磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000-NH2)得到DSPE-PEG2000-Folic，考察不同磷脂种类、药脂比、水化介质和载药时间，对脂质体粒径、包封率和稳定性的影响，确定脂质体的处方工艺。以大鼠的脑毛细血管内皮细胞(bEnd3)和星形胶质细胞组成体外血脑屏障(blood-brain barrier，BBB)，并结合大鼠胶质瘤C6细胞，构建体外模拟胶质瘤靶向治疗的复合BBB模型。考察阿霉素脂质体在bEnd3细胞中的摄取机制和透过BBB的转运速率及对C6细胞的毒性。结果 确定了DSPC作为主要磷脂组分，并以120 mmol·L^-1的硫酸铵作为水化介质，药脂比为1∶15，载药时间选择60 min，成功制备了高包封率和稳定性的双配体脂质体。其在bEnd3细胞中摄取远大于普通脂质体(P<0.05)，摄取过程受网格蛋白和小窝内陷介导的细胞内吞作用，并受转铁蛋白和叶酸的影响；同时其在BBB模型中的药物透过速率、及其进一步透过BBB后对下层C6细胞的毒性，均显著高于其他脂质体组。结论 转铁蛋白和叶酸共同修饰的阿霉素脂质体具有较好的体外脑胶质瘤靶向治疗作用。

Abstract:

OBJECTIVE To carry out the optimization of formulation and preparation process of the transferrin and folic acid co-modified doxorubicin liposomes [Tf (FA)-DOX-lipo], investigating the glioma targeting therapeutic effects. METHODS Tf (FA)-DOX-lipo was prepared by thin-film evaporation method and ammonium sulfate gradient method. First, folic acid was connected to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 to get the DSPE-PEG2000- Folic. Further, the phospholipid species, ratio of drug to lipid, hydration medium concentration and drug loading time were examined, particle size, encapsulation efficiency and stability were utilized to determine the formulation and process of the liposomes. Rats brain blood capillary endothelial cells (bEnd3) and astrocytes were used to construct in vitro blood-brain barrier(BBB), combined with rats glioma C6 cells to construct the composite BBB model, investigating glioma targeted therapy in vitro. The uptake mechanism of doxorubicin liposomes in bEnd3 cells, transport ratios through the BBB and toxicity on C6 glioma cells were investigated. RESULTS The results of NMR showed that folic acid was successfully connected to the DSPE-PEG2000-NH₂, while identifying DSPC as the main components of phospholipids, 120 mmol·L^-1 ammonium sulfate as the hydration medium, the drug to lipid ratio was 1∶15, drug time was 60 min, and stable dual-ligands modified liposomes with high encapsulation efficiency was successfully prepared. BEnd3 cell uptake was much larger than conventional liposomes (P<0.05), which demonstrated the brain targeting mechanism of Tf(FA)-dox-lipo were likely clathrin-dependent and caveolae dependent endocytosis pathway, subjected to the impact of transferrin and folic acid as well. In the BBB model, drugs transport ratio and toxicity on C6 cells after transport were significantly higher than other liposome groups. CONCLUSION Transferrin and folic acid modified doxorubicin liposomes have good glioma targeting and therapeutic effect in vitro.