面包酵母β-葡聚糖颗粒负载甲氨蝶呤抗急性单核细胞白血病的体外研究

In Vitro Study on BBG/MTX/Alg against Acute Monocytic Leukemia

目的:探讨面包酵母β-葡聚糖颗粒负载甲氨蝶呤缓释药物(以下简称:BBG/MTX/Alg)抗急性髓系白血病M5型(acute myeloid leukemia-M5,AML-M5)的效果。方法:采用溶解-沉淀法制备BBG/MTX/Alg,利用紫外分光光度计测定载药量,通过透射电子显微镜进行形貌表征,X射线粉末衍射反映负载情况,并在体外模拟胃肠道的释放条件测定缓释效率。CCK-8法检测药物细胞毒性及安全性。采用流式细胞仪检测各组凋亡和周期的比例。结果:MTX成功载入BBG空腔,负载量为7.8%,2 h时胃液模拟液中MTX释放率小于15%,24 h时在肠液模拟液释放率约95%。与生理盐水对照组相比,BBG/MTX/Alg能有效抑制AML-M5细胞系增殖且具有时间及浓度依赖性,流式细胞术结果表明其可显著促进AML-M5细胞的凋亡(P<0.001),且阻滞细胞周期于G0/G1期(P<0.0001)。与等量游离MTX相比,BBG/MTX/Alg处理24 h后对正常细胞系毒性较小(P<0.05)。结论:BBG可以作为MTX的有效缓释载体,BBG/MTX/Alg可对AML-M5细胞起到抑制增殖、诱导凋亡及阻滞细胞周期的作用,并且对正常细胞系毒性较小,提示其或可作为有效的抗AML-M5临床口服药物缓释剂。

Objective: To investigate the effect of BBG/MTX/Alg[methotrexate(MTX) carried by baker’s yeast-derived β-glucan(BBG) embedded in alginate gel crossed by Ca2+(Alg), hereinafter referred to as BBG/MTX/Alg]on acute myeloid leukemia-M5(AML-M5). Methods:Dissolving-precipitating method was used to prepare BBG/MTX/Alg. The load of MTXwas determined by ultraviolet-visible spectrophotometry. Particle morphology was observed by transmission electron microscope. X-ray diffraction was used to observe the encapsulation of MTX. The efficiency of BBG/MTX/Alg release was measured by releasing MTXinin vitrosimulatedgastrointestinal tract. The cytotoxicity and safety of drugs were measured by CCK-8. Percentages of cells in different cycles and apoptotic cellswere measured by flow cytometry. Results: 7.8% of MTX was loaded into BBG cavities successfully. The release rate of MTX in simulated gastric fluid was less than 15% at 2 h, and was about 95% in simulated intestinal fluid at 24 h. Compared with the control group(saline), BBG/MTX/Alg inhibited the proliferation of AML-M5 cells effectively in a time-and dose-dependent manner. Flow cytometry results showed that it significantly promoted the apoptosis of AML-M5 cells(P<0.001), and arrested cell cycle at G0/G1 phase(P<0.0001). Conclusion: BBG could be used as an effective sustained-release carrier of MTX. BBG/MTX/Alg could inhibit proliferation, induce apoptosis, and block cell cycle of AML-M5 cells, which has low toxicity to normal cell lines, indicating that it could be used as an effective sustained-release oral anti-AML-M5 agent.