槲皮素与紫杉醇主动靶向共输送治疗非小细胞肺癌协同作用及分子机制

The chemotherapeutic drug with no targeting leads to the treatment failure in non-small cell lung cancer. The combination chemotherapy is an effective way to improve therapeutic effect of lung cancer. Traditional Chinese medicine has a unique advantage in combined therapy for cancer, and it is an effective way to improve the therapeutic effect of lung cancer by combining the active parts of Traditional Chinese Medicine with chemotherapy. Supported by the National Natural Science Foundation of China, our previous studies confirmed that quercetin and paclitaxel co-loaded nano-in-micro large porous microsphere possessed a synergistic effect and reducing toxicity in treatment of lung cancer cell, however, the mechanism of synergistic effect of quercetin and paclitaxel on lung cancer is still unclear. Based on our previous research works, which have been successfully constructed a quercetin and paclitaxel nanoparticles loaded in a large porous microspheres, this project is intended to optimize the preparation process accomplished by evaluating the drug-loading as optimization index, and according to taking the signal path of Ras/Raf/MAPK and PI3K/AKT/mTOR as a breakthrough point, the safety and targeting efficacy, as well as the synergistic efficacy and possible molecular mechanisms of quercetin and paclitaxel loaded in active targeting of nano-in-micro large porous microspheres were evaluated by Western blot、Real-time quantitative PCR and in vivo imaging from the molecular, cellular and whole animal level. The results of this project will provide a new route and idea for clinical use of accurate targeted nano-in-micro large porous microspheres for pulmonary drug delivery with quercetin and paclitaxel loaded as an adjuvant therapy drug for lung cancer.

化疗药物无靶向性是导致非小细胞肺癌治疗失败的主要原因，联合化疗能显著提高疗效，中药在联合化疗中具有独特的优势，将具有协同增效作用的中药活性成分与化疗药物共输送成为提高肺癌治疗效果的有效途径。前期在国家自然科学基金资助下，我们证实槲皮素与紫杉醇共载于肺吸入靶向纳米复合大孔微球对肺癌细胞具有联合增效、减毒作用，但二者共输送增效肺癌的作用机制目前仍不清楚。本项目拟对载槲皮素与紫杉醇肺吸入靶向纳米复合大孔微球进行工艺优化，以信号通路Ras/Raf/MAPK及PI3K/AKT/mTOR为切入点，从分子、细胞及整体动物水平，通过Western blot、Real-time quantitative PCR、活体成像等技术评价其安全性、肺内分布及靶向性，采用体外、体内方法阐明槲皮素与紫杉醇靶向共输送协同增效的分子机制，为槲皮素应用于紫杉醇临床辅助化疗、提高肺癌治疗效果提供新途径与思路。

槲皮素与紫杉醇联合应用有协同作用，验证槲皮素及紫杉醇对A549和A549/Taxol的细胞毒性并找到作用的最优比例发现PTX=2 µg/mL、QUE=16 µg/mL；QUE及PTX对细胞的毒性主要是细胞凋亡引起的，细胞凋亡比例为47.58%，两药联合作用的机制是引起线粒体膜电位发生显著性变，证明槲皮素及紫杉醇/壳聚糖靶向纳米粒两药协同并能逆转耐药作用，作用机制是通过信号通路Akt、ERK而发挥作用。成功构建西妥昔单抗靶向的槲皮素及紫杉醇壳聚糖纳米粒，偶联率为9.13%，载药量及包封率分别为13%及90%左右，具有缓释特性。紫杉醇及槲皮素靶向纳米粒联合给药对A549及A549/Taxol有显著地协同及逆转效果；槲皮素与紫杉醇肺吸入靶向纳米复合大孔微球的最佳制备工艺为喷雾干燥条件进口温度 130℃，旋风 50，泵速 3 mL/min，压缩空气流量301 L/h（高度25 mm），微球的外观呈三维球状；槲皮素与紫杉醇肺吸入靶向纳米复合大孔微球具有良好的肺部安全性、靶向性及肺内分布，槲皮素与紫杉醇靶向共输送协同体外及体内皆有增效作用，其作用机制为通过Akt和ERK的相关信号通路发挥作用，提出将槲皮素与紫杉醇共载于表皮生长因子受体介导的肺吸入靶向纳米复合大孔微球即通过制剂技术实现中西结合治疗，是提高NSCLC临床治疗效果的有效途径，是对肺癌靶向治疗形式的拓展，将传统中医药与现代纳米技术交叉融合，为中药新剂型开发及肺癌治疗提供了新思路与新方法。