超临界流体技术构建共载siRNA和紫杉醇的微镶纳多孔高分子微球及其在肺癌治疗中的应用

To overcome the multidrug resistance (MDR) in the lung cancer therapy and the shortcoming in the use of traditional methods to produce porous polymer microspheres (PPMs) for pulmonary drug delivery, we attempt to employ the supercritical fluid technology (SFT) to produce the PPMs with a nanoparticles (NPs)-inlaid structure to co-deliver siRNA and paclitaxel (PTX) for lung cancer therapy. The chitosan (CS) NPs containing siRNA will be prepared by complex coacervation in the first place, and then the siRNA-CS NPs will be encapsulated in the PTX-loaded PPMs by supercritical fluid antisolvent process, which could protect the activity of siRNA during the processes of drug loading and drug delivery. After the direct inhalation of the PPMs co-loaded with siRNA and PTX into the lungs, the pump gene will be silenced by siRNA, thus unlocking the barrier of cancer cells to drugs and overcoming the MDR. By optimizing the operating parameters, the PPMs which meet the requirements of pulmonary drug delivery and loaded with siRNA with a good maintenance of activity will be achieved; the physicochemical properties of the resulting PPMs co-loaded with siRNA and PTX will also be characterized. The biocompatibility of the PPMs will be evaluated at various levels of cellular, integrated and molecular; and studies on their medical functions, particularly the pharmacodynamics, will be carried out in detail to investigate the synergistic effects of siRNA and PTX and mechanism on overcoming the MDR.

为克服目前肺癌治疗存在多药耐药性以及传统方法制备肺部给药用多孔高分子微球存在的缺点，本项目拟利用超临界流体技术构建具有微镶纳结构的多孔高分子微球共载siRNA和紫杉醇用于肺癌的治疗。首先采用复凝聚法制备负载siRNA的壳聚糖纳米粒，然后利用超临界流体抗溶剂法制得siRNA以被壳聚糖纳米粒包覆的形式镶嵌在基质中负载紫杉醇的多孔高分子微球，以保护siRNA载药和给药过程中的活性；经吸入给药直达肺癌病灶，siRNA发挥干扰作用，沉默编码泵蛋白基因，从根本上解除癌细胞对药物的屏蔽，克服多药耐药性。通过优化制备工艺参数，使制得的多孔高分子微球符合肺部给药的要求且较好地保持siRNA的活性；对共载siRNA和紫杉醇的微球进行系统的理化性能表征。在细胞、整体以及分子水平评价微球的生物相容性，并对其医用功能性尤其是药效学进行详细的研究，初步探索siRNA和紫杉醇的协同作用机制以及克服多药耐药性的机理。

本项目利用超临界流体抗溶剂法制备共载化疗药物阿霉素和克服多药耐药性siRNA基因药物的微镶钠多孔聚乳酸微球用于肺癌治疗的研究。.首先，探究了不同工艺参数对空白聚乳酸多孔微球的影响，所得最优工艺条件为：压力8 MPa，温度30℃，乳液流速4.0 mL/min，油相浓度2.3%，二氧化碳放气速率1000 L/h，水油比1/10。在此基础上，对高分子多孔微球分别利用胰岛素和甲氨蝶呤进行了载药性能研究，研究结果表明具有良好的载药性能和药学功效。.其次，制备了微镶钠多孔微球并对其生物相容性进行评价。所制得的微球表面粗糙且有孔洞，分散性较好，具有较好的空气动力学性能，且纳米颗粒在微球分布均匀。生物相容性研究方面，在细胞水平采用MTT法考察细胞毒性、硝酸还原酶法考察NO释放量，结果表明微球具有良好的细胞相容性；分子水平上，采用胞质分裂阻滞微核试验考察双核细胞微核率，结果显示微球具有良好的分子相容性；整体水平上，肺损伤实验结果证明，结果表明微球不会引起肺部的损伤。.最后，以靶向p糖蛋白的基因作为逆转耐药性的基因，使用壳聚糖对其进行包封，同时以阿霉素为化疗药物，使用超临界抗溶剂法制备共载基因和化疗药物的微镶钠多孔微球。采用MTT法考察不同组别对细胞的毒性，结果显示载药微镶钠多孔微球具有比单载药物的多孔微球及裸药具有更强的抗癌效果，细胞摄取实验显示，与裸药相比，微镶钠多孔微球远高于耐药细胞内的药物成分，吞噬实验证明纳米颗粒能够从微球中释放出来，并通过溶酶体逃逸发挥逆转多药耐药的作用。.本项目采用超临界流体技术成功地制备了共载化疗药物和基因的微镶钠多孔高分子微球，有潜力作为肺部给药剂型在化疗药物联合基因治疗领域发挥积极的作用。.在本项目的资助下，发表SCI论文20篇，其中一区和二区论文18篇。