犬肾上皮细胞对泊那替尼纳米混悬剂的摄取机制研究

摘要

目的 探讨犬肾上皮细胞(MDCK细胞)对泊那替尼纳米混悬剂的摄取机制.方法 采用溶剂蒸发法制备泊那替尼纳米混悬剂.高效液相法检测泊那替尼,评估其专属性、精密度及泊那替尼回收率;检测多药耐药蛋白(MDR)1抑制剂凡德他尼对泊那替尼纳米混悬剂细胞毒性作用的影响;采用高效液相法检测表面活性剂和凡德他尼对MDCK细胞摄取泊那替尼能力的影响.结果 采用高效液相法检测泊那替尼时,细胞溶液对检测无干扰,专属性强;日内和日间相对标准偏差分别为0.9％、1.2％,精密度良好;50、100、200 mg/L泊那替尼标准品回收率分别为101.7％、98.2％、102.4％,回收率高.细胞毒性实验结果表明细胞存活率随着泊那替尼纳米混悬剂浓度增加而降低;各泊那替尼浓度下,细胞存活率均随凡德他尼浓度增加而降低.MDCK细胞对泊那替尼摄取量随泊那替尼纳米混悬剂浓度的增加呈非线性增加.不同泊那替尼浓度下,MDCK细胞对含有表面活性剂的泊那替尼纳米混悬剂中泊那替尼的摄取量均高于对原料药中泊那替尼的摄取量[10 mg/L:(5.2±1.3) μg/mg蛋白比(3.0±0.8)μg/mg蛋白,20 mg/L:(6.6±2.3)μg/mg蛋白比(4.1±1.9) μg/mg蛋白,50 mg/L:(19.1±1.6) μg/mg蛋白比(7.3±1.7) μg/mg蛋白,100 mg/L:(30.3±1.0)μg/mg蛋白比(17.9±1.8) μg/mg蛋白,200 mg/L:(70.7±4.2)μg/mg蛋白比(53.6±5.3) μg/mg蛋白,400 mg/L:(94.7±3.4) μg/mg蛋白比(82.6 ±4.2)μg/mg蛋白](P＜0.05).4 mg/L和8 mg/L凡德他尼可明显增加细胞对泊那替尼纳米混悬剂中泊那替尼的摄取量[(4.65±0.11)、(5.70±0.24) μg/mg蛋白比(3.21±0.16) μg/mg蛋白,P＜0.05、P＜0.01].结论 MDCK细胞对泊那替尼纳米混悬剂的摄取可能受MDR1的介导.%Objective To investigate the mechanism of absorption of ponatinib nanosuspensions (PON-NPs) via madin-darby canine kidney (MDCK) cells.Methods PON-NPs were prepared by solvent evaporation technology.The specificity,accuracy and recovery of high performance liquid chromatography (HPLC) in detecting ponatinib (PON) were evaluated.The influence of multi-drugs resistance(MDR) 1 protein inhibitor and vandetanib on cell toxicity of PON-NPs was investigated.The influence of surfactant and vandetanib on the absorption of PON via MDCK cells was assessed.Results There was a strong specificity in detection of PON by HPLC,with no interference caused by cell solution;the within day precision and day to day precision were good with relative standard deviation of 0.9％ and 1.2％;the recovery rate was 101.7％,98.2％ and 102.4％ for 50,100,200 mg/L of PON standard substance,respectively.The cell survival rate was decreased with an increase of PON-NPs concentration.In the same concentration of PON-NPs,the cell survival rate was decreased with increase of vandetanib concentration.The intake of PON increased nonlinearly with increase of PON-NPs concentration.The cellular uptake of PON for PON-NPs was significantly higher compared with that for bulk drug of ponatinib [10 mg/L:(5.2 ± 1.3) μg/mg protein vs (3.0 ± 0.8) μg/mg protein,20 mg/L:(6.6 ± 2.3) μg/mg protein vs (4.1 ± 1.9) μg/mg protein,50 mg/L:(19.1 ± 1.6) μg/mg protein vs (7.3 ±1.7) μg/mg protein,100 mg/L:(30.3 ± 1.0) μg/mg protein vs (17.9 ± 1.8) μg/mg protein,200 mg/L:(70.7 ± 4.2) μg/mg protein vs (53.6 ± 5.3) μg/mg protein,400 mg/L:(94.7 ± 3.4) pg/mg protein vs (82.6 ±4.2) μg/mg protein] (P ＜0.05).The cellular uptake of PON-NPs was significantly increased after treatment with 4 mg/L and 8 mg/L of vandetanib [(4.65 ± 0.11),(5.70 ± 0.24) μg/mg protein vs (3.21 ± 0.16) μg/mg protein,P ＜ 0.05 or P ＜ 0.01].Conclusion The absorption process of PON-NPs across MDCK cells is possibly mediated by MDR1.