光控合成载siRNA的蛋白质纳米粒子治疗肿瘤的研究

RNA interference (RNAi) technology has great potential in the treatment of many diseases including cancer. How to realize the effective delivery of siRNA in vivo is crucial for the application of siRNA in fundamental and clinical studies. Proteins, as attractive carriers for small interference RNA (siRNA) delivery, have high biocompatibility and excellent biodegradability. Currently, preparation of most protein-based nano-carriers for drug delivery usually requires complicated chemical reactions, which are usually under harsh conditions. Here, we propose to develop a protein-based nano-carrier for siRNA delivery using relatively mild photo-induced self-assembly. The UV exposure will reduce the disulfide bonds of proteins selectively, which results in partially unfolding the proteins and subsequently self-assembling protein-based nano-carriers. Furthermore, specific moieties will be added to the surface of these nano-carriers for active tumor targeting. The efficiency of the delivered siRNA on the down-regulation of the targeted protein and the inhibition of tumor growth will also be evaluated. Meanwhile, we will use micro telescope technologies to track the intracellular delivery of these nano-carriers by attaching quantum dots to the carriers’ surfaces. In addition, the dynamic change of the target protein’s expression will be monitored using living cell imaging technique. The development of the protein-based nano-carriers will not only advance the application of proteins as gene carriers, but also provide more thorough understanding on the mechanisms of the inhibition effects of siRNA.

RNA干扰技术在针对包括肿瘤在内的多种疾病的治疗中极具潜力，如何实现siRNA在体内的有效递送成为其在基础研究和临床应用中的关键问题。蛋白质作为siRNA载体具有生物相容性好、易修饰等很多天然优势，目前蛋白质纳米载体往往需要在复杂的化学体系和苛刻的反应条件下合成。本项目拟利用紫外光照还原蛋白质分子内二硫键、诱导蛋白质发生部分去折叠并进而自组装形成纳米粒子的研究基础上，探索将siRNA载入该蛋白纳米颗粒中，并将肿瘤靶向分子修饰在纳米表面，以实现对肿瘤的主动靶向治疗，并检测siRNA在体内外对靶基因的沉默和对肿瘤的抑制效果；同时，本项目还将利用量子点标记结合显微观察技术跟踪该纳米载体在活细胞内完整的活动轨迹，以及细胞内靶蛋白表达量的动态变化过程。该项目可为siRNA的体内递送提供一种绿色、易生物降解、具肿瘤靶向性的新型蛋白质纳米载体，且对于理解纳米载体中siRNA的作用机理有重要意义。

RNA干扰技术在针对包括肿瘤在内的多种疾病的治疗中极具潜力，如何实现siRNA在生物体内的有效递送成为其在基础研究和临床应用中的关键问题。蛋白质作为siRNA载体具有生物相容性好、易修饰等很多天然优势，然而，目前蛋白质纳米载体往往需要在复杂的化学体系和苛刻的反应条件下合成。本项目在利用紫外光照还原蛋白质(溶菌酶)分子内二硫键、诱导蛋白质发生部分去折叠并进而自组装形成纳米粒子的研究基础上，将siRNA载入该蛋白纳米颗粒中，并将肿瘤靶向分子cRGD多头修饰在纳米表面实现了对肿瘤的主动靶向治疗，结果表明siRNA-溶菌酶-RGD作用组的老鼠，其肿瘤得到了有效抑制，抑制率达到了85%；在本项目执行中，我们还合成了“量子点-蛋白质”复合纳米体系并结合显微观察技术跟踪该纳米进入细胞的方式及其在活细胞内完整的活动轨迹,并提出了相关机制；此外，我们还对酪氨酸介导蛋白质光还原及自组装进行了研究，结果发现酪氨酸也可以介导含硫键蛋白质的光还原，并使蛋白质进一步发生自组装，这一研究结果大大扩展了光还原技术应用于生物纳米技术领域的选材范围。本项目的执行为抗肿瘤药物的体内递送提供了一种方法简易，绿色易降解、具肿瘤靶向性的新型蛋白质纳米载体，且相关的研究结果对于进一步理解纳米载体中siRNA的作用机理具有重要意义。