基于介孔硅胶纳米粒的逆转多药耐药性siRNA/药物共传递体系的构建及其递送机制的研究

The multidrug resistance in cancer cells is the major obstacle for effective cancer chemotherapy. A novel approach for cancer drug resistance is to establish an efficient gene and drug co-delivery system, which provides the opportunity of knocking down genes involved in drug resistance and helping to restore the intracellular drug levels that required for induction of apoptosis and cytotoxicity. However, co-delivery gene and drug to cancer cells for enhanced chemotherapy efficacy has been rarely reported due to the lack of efficient co-delivery techniques. The mesoporous silica nanoparticles (MSNs), which have been widely studied for the medical and biological applications, are showed great potential for guest molecules co-delivery system because of the highly regular mesoporous structure and large pore volume. Different types of MSNs have been successfully prepared and characterized by our research group, and these MSNs were employed as a cancer cell-specific carrier to deliver various model drugs. The purpose of this subject is to develop a siRNA and drug co-delivery system based on pore enlarged MSNs, which would deliver Pgp siRNA as suppressor of pump cellular resistance and docetaxel as model drug simultaneously into cancer cells for synergistic reversal of multidrug resistance. This system may knock down the Pgp gene expression and restore the intracellular drug concentration. In this study, in vivo synergistic inhibition will be tested in a multidrug resistant xenograft model, and the intracellular trafficking will be investigated to demonstrate the cellular internalization and the intracellular release of DTX and siRNA. Moreover, immunoblotting, qPCR analysis, and multiple biopsies will be used to explore the mechanism of the reversal of multidrug resistance. It was expected that this study would solve the clinical problems of chemotherapy failure due to multidrug resistance by utilizing the novel MSNs as efficient co-delivery carriers for drug and gene in vivo applications.

多药耐药是肿瘤化学治疗的最大障碍，也是单纯药物治疗失败的主要原因之一。利用基因与化疗药物的协同作用，可特异性地沉默靶基因，抑制耐药蛋白表达，从而达到更好治疗效果。如何将基因和药物同时递送到肿瘤细胞是其发挥协同效应的关键。传统给药系统往往无法满足体内共传递的要求，因而构建高效共传递系统是目前基因与药物协同治疗迫切需要解决的难题。介孔硅胶纳米粒高度有序的孔道结构能够在理论上确保多种客体分子的装载。前期研究证实，该载体能够递送不同药物分子进入细胞。本项目拟针对肿瘤多药耐药生物学机制，构建基于扩孔介孔硅胶纳米粒的基因（p糖蛋白siRNA）和药物(多西紫杉醇)的共传递体系，保护siRNA的活性，辅助siRNA及药物进入肿瘤细胞发挥基因沉默效应及抗癌作用。并通过荧光标记-亚细胞器定位分析及免疫印迹法，从细胞及分子水平探讨介孔硅胶纳米粒共传递系统的递送机制，为基因与药物共传递系统的研究提供实验依据。

耐药是现阶段肿瘤治疗领域尚未解决的一大难题。研究表明，药物通过耐药相关蛋白的外排被认为是癌细胞产生耐药现象的重要原因。将靶向编码相关蛋白基因的siRNA递送进入细胞可使对应外排蛋白表达下调，从而降低细胞对化疗药物的外排作用，提高治疗效果。如何将基因和药物共同递送到肿瘤细胞是其发挥协同效应的关键。传统给药系统往往无法满足体内共传递的要求，因而构建高效共传递系统是目前基因与药物协同治疗迫切需要解决的难题。为此，我们探索构建了基于新型无机材料介孔硅胶纳米粒的基因和药物共传递体系用于解决肿瘤耐药问题。首先，为了满足共传递体系的不同递送需求，我们成功制备出多种不同结构特征和靶向配体修饰的介孔硅胶纳米粒，并进行了相关表征。然后，成功将化疗药多西紫杉醇装载于乳糖配体修饰的介孔硅胶纳米粒，优化体系的载药量及药物释放性质，试验结果表明该体系对癌细胞具有显著的杀伤效果。然后，我们研究了介孔硅胶纳米粒的基因吸附行为，结合载体性质，研究了配体修饰及吸附条件对载体基因装载性能和装载稳定性的影响，制备了装载量高的基因载体复合物，为提高载体的基因装载性能提供了重要参考。进一步，对递送体系的基因转染效果进行评价，结果表明该递送体系在提高转染效率的同时提高了载体的生物安全性。最后，对基因冻干制剂的稳定性进行了全面深入的研究。经过处方筛选，制备了稳定性良好的介孔硅胶基因冻干制剂，对基因制剂的的贮存、运输以及使用均具有重要意义。本项目发表了多篇药剂学SCI论文，在国内外被广泛引用。本研究拓展了介孔硅胶材料在基因与药物递送领域的应用，为高效安全基因递送体系的构建提供了重要的理论指导和实践价值。