胡桃醌-PLGA纳米粒制备及对A375恶黑细胞的体外抗肿瘤作用

目的:制备胡桃醌(juglone,Jug)聚乳酸-羟基乙酸(poly lactic-co-glycolic acid,PLGA)纳米粒(Jug-PLGA-NPs),并考察其理化性质、体外释放特征及对A375细胞的体外影响。方法:采用乳化挥发法制备Jug-PLGA-NPs,对其粒径、包封率、载药率以及体外释放特征进行考察;荧光显微镜观察PLGA-NPs在体外细胞的摄取情况,小动物活体成像仪观测PLGA-NPs在BALB/c荷瘤裸鼠尾静脉注射后体内的分布;用噻唑蓝(thiazolyl blue tetrazolium bromide,MTT)比色法检测其对A375细胞增殖抑制作用,流式细胞仪进行细胞凋亡率及细胞周期检测;蛋白免疫印迹法检测蛋白激酶B(protein kinase B,Akt),磷酸化-Akt(p-Akt),周期蛋白D1(cyclin D1)的表达情况。结果:制备的Jug-PLGA-NPs平均粒径为(149.6±21.5) nm,包封率为(68.39±2.51)%,载药率(5.07±0.98)%,具有良好的缓释特征。PLGA-NPs在体外细胞摄取和体内活体成像中具有良好的穿透和靶向性能。不同浓度Jug-PLGA-NPs均能明显抑制A375细胞增殖、促进细胞凋亡,呈明显时间浓度依赖性(P0.05),且48 h作用略优于等浓度Jug;其机制可能与调节Akt磷酸化水平,下调cyclin D1表达(P0.05),阻滞细胞于G0/G1期有关(P0.05)。结论:负载Jug的PLGA纳米微粒制备简便,具有良好的药物缓释、肿瘤靶向及抗肿瘤能力,为未来Jug的临床应用提供了一种新的药物剂型。

Preparation of Juglone-loaded PLGA Nanoparticles and Its Anti-tumor Effect on Melanoma A375 Cells in Vitro

Objective: To prepare Juglone-loaded poly lactic-co-glycolic acid nanoparticles (Jug-PLGA-NPs), and investigate their physicochemical properties, release characteristics in vitro and anti-tumor activities on A375 melanoma cells in vitro. Method: Jug-PLGA-NPs were prepared by emulsification-solvent evaporation method. Then the particle size, encapsulation efficiency, drug loading rate and in vitro release characteristics were investigated. Fluorescence microscopy was used to observe the uptake of PLGA-NPs in vitro. The distribution of PLGA-NPs in BALB/c nude mice after tail vein injection was observed by the small living animal imaging system. Their inhibition effect on proliferation of A375 cells was detected by thiazolyl blue tetrazolium bromide (MTT) assay. Apoptosis rate and cell cycle detection were performed by flow cytometry. Western blot was used to determine the protein kinase B (Akt), phosphorylated Akt (p-Akt) and cyclinD₁. Result: The average particle size of the prepared Jug-PLGA-NPs was (149.6±21.5) nm, entrapment rate of (68.39±2.51)%, and drug-loading rate of (5.07±0.98)%, showing good sustained-release characteristics. PLGA-NPs showed good penetration and targeting properties in cellular uptake in vitro and in vivo imaging. Different concentrations of Jug-PLGA-NPs could significantly inhibit the proliferation and promote apoptosis of A375 cells in a time and concentration dependent manner (P<0.05), and its 48 h effect was superior to that of the same concentration of Jug. The mechanism may be related to the regulation of Akt phosphorylation level, down-regulation of cyclinD₁ expression (P<0.05) and the cell-cycle arrest in G₀/G₁ phase (P<0.05). Conclusion: The Jug-PLGA-NPs are easy to prepare and have good sustained-release characteristics, tumor targeting and anti-tumor ability, providing a new pharmaceutical dosage form for the future clinical application of Jug.