基于新型给药系统的槲皮素与紫杉醇联用抗肺癌增效作用机制研究

The bottleneck of chemotherapeutic failure of lung cancer is that the chemotherapeutic drugs have serious toxic side effects and without targeting to cancer, anti-tumor traditional Chinese medicines which can clear away heat and toxic material, and strengthening vital qi to eliminate pathogenic factor have satisfactory efficacy against lung cancer in clinic, which most of them contain the ingredient of quercetin. Quercetin possesses the effects of strong anti-oxidant and anti-tumor, as well as synergistic effects in combination with paclitaxel, the first-line chemotherapeutic drug. But quercetin shows poor water solubility and low bioavailability, which limits its clinical use in cancer therapy. According to the advantages of nanoparticles, the active components of traditional Chinese medicine loaded in nanoparticles can significantly improve the physical and chemical characteristics of drug and the bioavailability. Based on our previous study, we hypothesized that target nano-in-large porous microsphere as a pulmonary drug delivery system co-deliverying quercetin and paclitaxel is an effective method to improve the effect in treatment of lung cancer. This project is intended to develop a nano-in large porous microsphere with quercetin and paclitaxel loaded targeting to epidermal growth factor receptor as pulmonary drug delivery system, further to explore the synergistic efficacy and possible molecular mechanism by applying Ras/Raf/MAPK singnal pathway on cell and animal model. The results of this project will provide certain experiment’s evidence and theoretical basis for the clinical use of nano-in large porous targeting microspheres for pulmonary drug delivery system with quercetin and paclitaxel loaded as an adjuvant therapeutic drug in lung cancer.

针对肺癌化疗药物没有靶向性及严重的毒副作用导致化疗失败的瓶颈，抗肿瘤的清热解毒、扶正祛邪类中药临床上对肺癌疗效确切，此类中药多数含有槲皮素，该成分具有抗氧化、抗肿瘤功效，与一线化疗药物紫杉醇联用具有协同增效作用，但其水溶性差，生物利用度低。基于纳米粒能改善药物性质、明显提高生物利用度，结合前期研究工作基础，提出假设：将槲皮素及紫杉醇共载于肺吸入靶向纳米复合大孔微球可能是提高肺癌治疗效果的有效途径。拟构建表皮生长因子受体（EGFR）靶向的共载槲皮素与紫杉醇肺吸入纳米复合大孔微球，以Ras/Raf/MAPK 信号通路为切入点，从体内、体外水平共同探讨槲皮素与紫杉醇肺吸入靶向纳米复合大孔微球靶向肺癌的治疗作用，验证联合用药的功效及槲皮素与紫杉醇联合的协同增效作用，揭示协同作用的分子机制，为难溶性中药有效成分槲皮素应用于紫杉醇临床辅助化疗、构建肺癌治疗的新型给药系统提供必要的实验依据和理论基础。

以油酰壳聚糖为载体分别制备槲皮素/油酰壳聚糖纳米粒和紫杉醇/油酰壳聚糖纳米粒，采用喷雾干燥法制备载槲皮素及紫杉醇纳米粒的肺吸入纳米聚合大孔微球。槲皮素/油酰壳聚糖纳米粒及紫杉醇/油酰壳聚糖纳米粒外观大小均一，呈类球形，分散均匀；粒径均小于300 nm，多分散性指数（PDI）小于0.300，zeta电位在+25 mV左右。槲皮素/油酰壳聚糖纳米粒和紫杉醇/油酰壳聚糖纳米粒中的槲皮素和紫杉醇的载药量分别为14.15%和15.01%，包封率分别为44.60%和48.80%，具有缓释作用。肺急性毒性表明载药纳米粒对肺无毒性。槲皮素和紫杉醇共输送肺吸入油酰壳聚糖纳米聚合大孔微球呈球形，微球分散均匀，粒径为3373 nm，PDI为0.456，载药量分别为14.79%和15.36%，包封率分别为90.30%和92.60%。能够分散为小于200 nm左右的纳米颗粒。槲皮素与紫杉醇纳米聚合微球在磷酸缓冲液（PBS，pH 7.4和pH 4.5）中具有缓释作用，直到48 h，槲皮素和紫杉醇在pH 7.4的PBS缓冲溶液中累计释放量分别为60.88%和52.40%，而pH 4.5时，槲皮素和紫杉醇累计释放量分别为70.85%和60.05%。体内药动学结果显示联合用药比单独用药时提高了紫杉醇在体内的血药浓度，肺部给药6 h后仍然能使药物在肺部维持较高的浓度。MTT说明紫杉醇与槲皮素共给药对肿瘤细胞的生长抑制有协同作用（63.0%，A549；69.7%，H1299），而是紫杉醇与槲皮素共给药发挥的协同作用导致的，也与细胞迁移实验的结论相吻合。这些结果有望解决局部化疗药物浓度过低导致的化疗失败的瓶颈，为槲皮素增敏紫杉醇联合化疗提供新剂型及新思路。