具有肿瘤双重靶向及内涵体逃逸功能的高siRNA载量纳米粒的制备及形成机制研究

An ideal anti-tumor effect is not yet obtained even if siRNA has been delivered into target cells, which is believed related to the degradation of siRNA by acid and enzymes in endosomes/lysosomes and insufficient siRNA for gene silencing in cytoplasm. So, it is crucial to deliver sufficient siRNA into cytoplasm to realize gene therapy. The presence of siRNA conglomerations, which are composed by almost entire siRNA, shows hope for sufficient siRNA in cytoplasm. Unfortunately，the efficient siRNA delivery of this system into cytoplasm is invalidated by lack of tumor targeting and thereafter endosomal degradation. To acquire satisfactory gene therapy，dual tumor-specific targeting nanocarriers with high siRNA loading and endosomal escape ability (DTNHLE) will be constructed by combining siRNA polymerization with ligands and PEG decorated polyethyleneimine (YIGSR/PHSCNK-PEG-PEI). These nanocarriers will get tumor sites under the guidance of dual targets in vivo, then be internalized by tumor cells because of the high affinity between receptors and ligands and transported into endosomes. Polyethyleneimine，a kind of cationic polymers, will be subsequently liberated after degradation of YIGSR/PHSCNK-PEG-PEI by acid and enzymes in endosomes and destruct the membrane of endosomes via proton sponge effect. siRNA polymers are uncaged into cytoplasm and sufficient of siRNA is released in cytoplasm under the action of Dicer. DTNHLE will be constructed by using siRNA polymerizing, long-circulating, dual tumor-specific targeting and endosomal escaping technology combinedly. The formation mechanism of DTNHLE will also be investigated to obtain the optimal DTNHLE. Sufficient siRNA delivery into cytoplasm of tumor cells and desirable inhibition of tumor are expected in this study.

如何显著提高靶细胞浆内水平是限制siRNA临床应用的关键所在。siRNA聚合体由纯siRNA组成，克服了现有载体siRNA载量低的缺陷，成为siRNA临床应用的希望所在。然而，非特异性及内涵体降解作用导致其到达靶细胞浆内的siRNA水平有限。本课题拟将配体及PEG修饰的阳离子聚合物PEI与siRNA聚合体复合，构建具有长循环靶向及内涵体逃逸能力的高siRNA载量纳米粒（DTNHLE）。DTNHLE将在配体PHSCNK肽和YIGSR肽双重介导下靶向肿瘤组织，并经内吞高效透膜，在内涵体中酸及酶作用下解离出PEI，获得内涵体逃逸能力，siRNA聚合体则在胞浆Dicer酶作用下释放大量活性siRNA，实现靶细胞浆内siRNA水平的显著提高。本项目将综合运用siRNA聚合技术、长循环修饰技术、双重靶向技术以及内涵体逃逸技术，探索构建DTNHLE的可能性及其形成机制，为推动其临床应用奠定基础。

siRNA是肿瘤治疗领域最有前景的药物之一，但其体内降解速度极快，即使进入靶细胞浆亦难以达到有效剂量，故提高siRNA在靶细胞浆内的水平势在必行。siRNA聚合体是siRNA单元的高度集合，全部由RNA序列组成，代替单个siRNA分子包裹于载体中，可克服现有载体siRNA载量低的问题，将高载量与现有载体的长循环靶向等功能有效结合，有望获得理想的治疗效果。本研究探索了RNA聚合体的转录方法，构建了PEI-壳聚糖长循环靶向纳米粒，并对其进行了表征与评价。主要研究结果包括：（1）建立了siRNA聚合体的制备方法：建立了包括单链环状模板转录、单链环状模板纯化后转录、双链环状模板转录、双链环状鼓泡模板转录、调整不同序列结构进行双链转录、linker集中双链环状转录方法等在内的六套siRNA转录方法，比较了不同方法的转录效果差异，系统考察了包括转录酶、转录条件以及转录模板等关键因素对体外滚环转录效率的影响，最终确定采用linker集中双链环状转录法制备siRNA聚合体，所获得的siRNA聚合体分子量最高为2000bp，纯化后浓度为96.5-118.73µg/ml。（2）构建了载siRNA聚合体的长循环靶向纳米粒：建立了通过静电相互作用制备纳米粒的方法，并考察了壳聚糖分子量、PEI分子量、壳聚糖浓度以及三聚磷酸钠的加入量等关键因素对纳米粒粒径的影响，确定了最优处方工艺；最终获得的纳米粒为圆形或类圆形，粒径约为180nm，Zeta电位约为27 mV，其于室温条件下放置72h稳定性。（3）siRNA聚合体纳米粒的评价：体外细胞评价结果显示，相对于无靶向修饰以及单靶向修饰的载体，双靶向修饰的纳米粒具有更强的细胞毒性、更强的胞内摄取能力、更显著的细胞凋亡率；小鼠体内抑瘤实验结果显示，双靶向修饰的纳米粒具有最强的肿瘤抑制效果，抑瘤率达到58.86%，且研究过程中未发现急性或严重毒性出现。