CAP1-siRNA靶向纳米载体在转移性肺癌中的作用机制研究

Our present study find that adenylate cyclase-associated protein 1 (CAP1) is connected with the metastasis of lung cancer. siRNA-based RNA interference (RNAi) is a post-transcrip-tional process triggered by the introduction of double-stranded RNA (dsRNA) which leads to gene silencing in a sequence-specific manner. Using this advanced technology, we will make a novel class of proton-sponge coated Quantum dots ( QDs ), PMAL grafted with PEI loaded with CdSe/ZnSe QDs will be prepared through direct ligand-exchange reactions and surface modifications as multifunctional complexes for siRNA delivery and real-time intracellular imaging. The dual modality of QD-siRNA complexes allows real-time tracking of QDs and siRNA release during siRNA delivery. The unique properties of proton-sponge coated QDs can be further optimized in vivo for tumor targeting, drug delivery and simultaneous imaging due to their high stability and superior silencing efficiency in serum-containing environments. These modifications have the potency to improve siRNA stability as well as pharmacokinetic properties of the molecule. Overall, we are optimistic that chemical modification in addition to conjugation strategies for various biomolecules will play a pivotal role in the transition of siRNA from a laboratory knock-down tool into a valuable platform for the development of therapeutics. Currently, however, studies on the correlation between CAP1 expression and tumor metastasis-mediated signaling pathway are scarce. Nevertheless, this requires further evaluation.

肺癌已成为威胁人类生命健康的发病率和死亡率最高的恶性肿瘤之一。在前期国家自然基金资助下首次发现高转移肺癌中腺苷酸环化酶关联蛋白1(cyclase-associated protein 1,CAP1)的表达比正常组织高，高表达的CAP1与肺癌转移相关，由此推测CAP1与肺癌转移的发生发展有关。本项目将在此研究基础上进一步应用新型细胞-SELEX方法筛选制备高转移肺癌细胞A549的靶向适配体，并研发不同细胞穿膜肽共同修饰自带荧光量子点的新型纳米载体，有效实现穿越肺癌转移组织细胞的靶向途径。并且针对高表达CAP1转移肺癌的治疗，将构建的量子点靶向显像诊断载体系统高效负载绿色荧光标记的CAP1-siRNA，应用此RNAi干扰技术下调肺癌转移瘤细胞中的CAP1，并对其生物学改变和对肺癌转移动物模型的影响进行研究。这将对明确肺癌转移的机制打下理论基础，亦对肺癌转移早期诊断、提高靶向疗效等有着重要意义。

本研究将在之前的研究基础上，进一步应用新型细胞-SELEX方法所筛选制备出的对应转移性肺癌细胞A549的靶向适配体，并研发适配体及荧光标记共同修饰的新型纳米载体，有效实现穿越转移性肺癌肿瘤细胞的靶向运输途径。并且针对高表达CAP1的转移性肺癌，构建具有靶向性的高效负载CAP1-siRNA的荧光标记纳米载体，应用RNAi干扰技术下调肺癌转移瘤细胞中的CAP1，从而针对转移性肺癌进行治疗，并对其产生的生物学改变和在肺癌转移动物模型中的作用进行研究。. 本研究成功合成了适配体，并验证了其靶向性；成功合成CAP1-siRNA，并证实其可下调CAP1表达；成功合成并表征CAP1-siRNA靶向纳米载体，证实其符合进一步实验要求。在细胞实验部分，证实了CAP1-siRNA靶向纳米载体在一定浓度内对细胞毒性低，可被肺癌A549细胞摄取，对肺癌细胞有一定的靶向性，可降低肺癌A549细胞CAP1表达，从而减低其侵袭和迁移能力。在动物实验部分证实了CAP1-siRNA靶向纳米载体可在动物的肿瘤组织中分布，并可降低肺癌组织中CAP1的表达，有一定的靶向性，可减低肺癌转移，且在一定范围内安全性良好。. 本研究成功构建了CAP1-siRNA靶向纳米载体，且该CAP1-siRNA靶向纳米载体在体内外均可减低肺癌CAP1的表达，并具有一定的靶向性。同时该CAP1-siRNA靶向纳米载体可降低肺癌细胞侵袭和迁移能力，从而抑制肺癌转移。这将对明确肺癌转移的机制打下理论基础，同时对肺癌转移早期诊断、以及靶向性疗效等具有重要意义。