胞内触发释药的仿生型siRNA自组装胶束纳米复合物的构建、抗肿瘤转移作用及细胞内药物动力学研究

Viruses are natural masterpieces of nucleic acid drugs with the stable structure outside the cell, disassembly and dynamic process as they enter living cells, which present a good enlightenment for the design of artificial carriers for synthetic nucleic acids such as siRNA. Based on bionic principles, a polymeric micellar system was constructed from degradable chitosan grafted with spermine (SP), Octyl and PEG by the disulfide bond in this project. The virus mimetic shell was conferred by attaching two ligands, i.e., the integrin αvβ3-specific ligand (cRGD) for active cancer targeting and the influenza virus haemagglutinin HA2 for membrane-disruption of endosomes. These structures contain elements that mimic the delivery functions of viral particles and surface domains that shield against undesired biological interactions and enable specific host cell receptor binding through the presentation of targeting ligands cRGD. After internalization by receptor-mediated endocytosis into the acidifying endosomes of cells, synthetic siRNA can escape from these vesicles through the activation of membrane-disruption domains of HA2 as viruses do and reach the cytoplasm, the location of RNA interference. The siRNA against VEGF gene was entrapped into this virus mimetic polymeric micellar system to improve the efficacy of siRNA against VEGF gene in human tumor models that over express integrin receptor αvβ3. Then the tumor metastasis obtained by tumor angiogenesis could be solved effectively. In addition, Fluorescence resonance energy transfer (FRET) was achieved between FITC-VEGF siRNA and PbS QDs in the polymeric micellar system. From confocal microscopic analysis, intracellular uptake and release of siRNA from the polymeric micellar system were visualized as a function of incubation time. The extent of siRNA release from the complex was quantitatively evaluated by flow cytometric analysis.

病毒是一种天然的核酸药物载体，其细胞外稳定存在，细胞内解组装且动态转染的特点为非病毒基因载体的设计提供了很好的启示。模仿病毒的结构，本项目构建了一种以壳聚糖为基本骨架，精胺和疏水长链辛基为核心、肿瘤靶向肽RGD和融合肽HA2共同修饰的仿生型胶束纳米载体。该载体具备主动靶向、内涵体逃逸、触发释药、长循环、安全稳定等多种性能。并且以靶向VEGF基因的siRNA为模型药物制备仿生型siRNA胶束纳米复合物，期望能解决siRNA在生物体内容易被降解，转染效率低的问题，增强靶向VEGF基因的siRNA抗肿瘤生长和转移的效应。同时本项目还利用量子点独特的光学特点以及荧光共振能量转移的作用，动态监测该复合物在细胞内摄取、分布、释放siRNA的动力学过程。预计本项目的实现将为研发安全高效的siRNA药物传输系统提供一种新的思路，并为细胞内药物动力学研究创立一种快速、准确的新方法。

本项目首先通过化学合成的方法制备RGD/HA2修饰、有腙键连接的仿生型纳米胶束（RGD-PEG-HA2-CS-octyl-Lys-9R (hydrazone)）。采用高效液相-质谱联用（LC-MS）、核磁共振氢谱（1H NMR）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）对合成的RGD-PEG-HA2-CS-octyl-Lys-9R (hydrazone)进行表征。并分别选用粒度仪和透射电镜对所构建的仿生型胶束纳米粒进行粒度测试和形貌分析。采用MTT实验对该载体的生物相容性进行评价。.然后，将制备得到的仿生型壳聚糖胶束纳米载体通过静电结合与靶向VEGF基因的siRNA结合形成siRNA胶束纳米复合物，并采用琼脂糖凝胶电泳对其结合能力进行表征。激光共聚焦显微镜观察siRNA胶束纳米复合物对肿瘤细胞的靶向作用、对肿瘤细胞的转染作用，Western Blotting实验观察其基因沉默作用，MTT法测试其抗肿瘤血管内皮细胞生长作用。.为了实时监测制备得到的仿生型壳聚糖胶束纳米载体释放siRNA的动力学过程，本研究制备了油溶性CdSe量子点，并将其包裹在壳聚糖纳米胶束的核心内，与量子点同时包裹的还有Cy3标记的siRNA，CdSe量子点与Cy3标记的siRNA之间产生FRET作用。胶束纳米载体中所含的腙键在肿瘤细胞内的低pH环境下逐渐断裂，FRET作用逐渐消失。FRET作用可通过荧光分光光度计进行检测，进而可动态监测胶束内siRNA的释放过程。.本研究结果显示，RGD-PEG-HA2-CS-octyl-Lys-9R (hydrazone)纳米胶束已成功制备，其具有均一的粒径分布（大约215 nm）、较好的形貌特征、良好的生物相容性，显著的肿瘤细胞靶向作用，与siRNA结合后能显著提高转染效率、增强基因沉默作用及抗肿瘤血管内皮细胞生长作用。项目设计的siRNA胶束纳米载体充分利用阳离子聚合物壳聚糖易于修饰的优点，制备出具有肿瘤靶向、内涵体逃逸、胞内触发释放、长循环、高效稳定等多种性能的仿生型非病毒基因载体-壳聚糖纳米胶束。其成功构建将为研发安全高效的siRNA药物体内传输系统提供一种新的思路。另外，本研究还初步建立了通过FRET效应实时监测纳米载体动态释放siRNA的方法。