甲氨喋呤长循环脂质体对人骨肉瘤细胞的体外抗瘤活性

Long-circulating methotrexate-loaded liposomes exhibit an antitumor effect on human osteosarcoma in vitro

背景:脂质体作为一种新型给药系统,具有不良反应小、无免疫原性和可生物降解等优点,将甲氨喋呤包载入脂质体中可显著降低药物毒性,改善药物抗肿瘤效果。目的:制备甲氨喋呤长循环脂质体,考察其对人骨肉瘤细胞MG-63的抗瘤活性。方法:采用薄膜分散法制备传统甲氨喋呤脂质体,后插入法制备甲氨喋呤长循环脂质体(甲氨喋呤初始质量浓度分别为9.1,1.82,0.364 g/L),分别采用超速离心法、葡聚糖凝胶G-10微柱离心法和葡聚糖凝胶G-50微柱离心法去除甲氨喋呤长循环脂质体未包载的游离药物,对3种方法纯化后脂质体的回收率、粒径、形态、包封率及药脂比等理化性质进行表征。分别以0,1,5,25 mg/L甲氨喋呤长循环脂质体(选择超速离心法与葡聚糖凝胶G-50微柱离心法纯化的脂质体)和游离甲氨喋呤稀释液培养人骨肉瘤细胞MG-63,24,48 h后采用MTS法检测细胞毒性。结果与结论:(1)甲氨喋呤长循环脂质体的粒径在200 nm左右,为均匀的球形或近球形;(2)回收率实验结果显示,葡聚糖凝胶G-50微柱离心法是最合适的甲氨喋呤长循环脂质体纯化方法,具有回收完全和快速的优点;(3)甲氨喋呤初始质量浓度相同时,超速离心法、葡聚糖凝胶G-10微柱离心法纯化的脂质体包封率与药脂比明显低于葡聚糖凝胶G-50微柱离心法纯化的脂质体;(4)体外细胞毒性实验表明,在同质量浓度条件下,甲氨喋呤长循环脂质体的细胞毒性远高于游离甲氨喋呤,葡聚糖凝胶G-50微柱离心法纯化的甲氨喋呤长循环脂质体细胞活性普遍比超速离心法纯化的脂质体细胞毒性高;(5)结果表明,甲氨喋呤长循环脂质体对于人骨肉瘤细胞具有较强的体外抗瘤活性。

BACKGROUND： Liposomes as a new drug delivery system are characterized by few adverse reactions no immunogenicity and biodegradation. Furthermore, methotrexate-loaded liposomes can significantly reduce drug toxicity and improve anti-tumor effect. OBJECTIVE： To prepare long-circulating methotrexate-loaded liposomes and to evaluate its antitumor activity in MG-63 in vitro. METHODS： The iposomes were prepared using the film dispersion method, and the long-circulating ones were prepared using the post-insertion method. The initial concentrations of methotrexate were 9.1, 1.82, 0.364 g/L. The ultracentrifugation method and spin column method with Sephadex G-10 or G-50 as packing were employed to separate free drugs from the methotrexate-loaded liposomes. Their recovery morphology, encapsulation efficiency and drug-to-lipid ratio were evaluated. The cytotoxity of the size long-circulating methotrexate-loaded liposomes purified with ultracentrifugation method and spin column G-50 method under three dose levels （0, 1, 5, 25 mg/L） were determined by MTS method. RESULTS AND CONCLUSION： According to the recovery rates of three methods, the spin column G-50 method was considered as optimal for the long-circulating methotrexate-loaded liposomes. The long-circulating liposomes were spherical or ellipsoidal under transmission electron microscope, about 200 nm in size. At the certain initial concentration of methotrexate, the encapsulation efficiency and drug-to-lipid ratio of the liposomes purified using the spin column G-50 method were remarkably higher than those purified using the other two methods. At the same mass concentration, the cytotoxity of the liposomes purified with ultracentrifugation or spin column G-50 was significantly higher than that of free methotrexate, and furthermore, the cytotoxity of the liposomes purified with spin column G-50 was higher than that of the liposomes purified with ultracentrifugation method. To conclude, the long-circulating methotrexate-loaded liposomes show a higher antitumor activity than free methotrexate in MGo63 cells in vitro, providing the basis for further investigation of its antitumor effect on human osteosarcoma in vivo.