Novel nanoparticles for siRNA delivery

描述（由申请人提供）：近年来，我们已经成功地修改了脂质体/鱼精蛋白/DNA （LPD）纳米颗粒配方，用于在异种移植模型中靶向递送siRNA到人肺癌细胞。这个核/壳，自组装的纳米颗粒包含一个紧凑的鱼精蛋白/DNA/siRNA核，由两个阳离子脂质双层包裹。当纳米颗粒与聚乙二醇(PEG)-磷脂缀合物（DSPE-PEG）孵育时，外层双分子层被剥离，但内层双分子层因高度的聚乙二醇化而存活下来。密集堆积的表面聚乙二醇形成了一个刷状保护层，屏蔽了纳米粒子的阳离子电荷，减少了血清蛋白的调控作用。结果是肝脏和脾脏的摄取程度非常低，而肿瘤的摄取水平非常高，每g组织的注射剂量高达60-80%。如果一个靶向配体，异氨酰胺，被拴在PEG的远端，靶向纳米颗粒有效地传递siRNA来沉默整个肿瘤中的靶基因。然而，仔细观察递送的siRNA，大部分剂量被隔离在核内体中，不具有生物利用性。这是因为该制剂不具有任何内体溶解活性。因此，我们建议用磷酸钙（CaP）取代LPD的核心，磷酸钙在酸性核内体ph下溶解。我们预计溶解的CaP会显著增加核内体的渗透压，诱导细胞器肿胀和破裂，导致被封装的siRNA释放。提出的非病毒载体的改进将结合高水平的肿瘤摄取和有效的内体释放siRNA货物。该项目的另一个目标是治疗血管不那么渗漏的肿瘤。我们成功地将siRNA和阳离子脂质DOTAP混合在氟烃溶剂中，在高温高压下制备了小型生物活性脂质体（SBL）。所得纳米颗粒体积小（30-50 nm），转染活性强。我们将进一步对SBL表面进行聚乙二醇化和配体系聚修饰。我们期望新的纳米颗粒将siRNA传递到肿瘤细胞中，在肿瘤细胞中，新生血管没有那么多渗漏。

DESCRIPTION (provided by applicant): In the recent years, we have successfully modified the Liposome/Protamine/DNA (LPD) nanoparticle formulation for targeted delivery of siRNA to the human lung cancer cells in a xenograft model. This core/shell, self-assembled nanoparticles contained a compact protamine/DNA/siRNA core which is wrapped around by two cationic lipid bilayers. When the nanoparticles were incubated with a polyethylene glycol (PEG)-phospholipid conjugate (DSPE-PEG), the outer bilayer was stripped off, but the inner bilayer survived with a high degree of PEGylation. The densely packed surface PEG formed a brush protection layer to shield the cationic charges of the nanoparticles and reduced opsonization by serum proteins. The result was a very low degree of uptake by the liver and the spleen, and a very high level of tumor uptake, up to 60-80% injected dose per g tissue. If a targeting ligand, anisamide, was tethered to the distal end of PEG, the targeted nanoparticles efficiently delivered siRNA to silence a target gene in the entire tumor. However, upon a closer look at the delivered siRNA, most of the dose was sequestered in the endosomes and was not bioavailable. This is because the formulation did not possess any endosomal lytic activity. Thus, we propose to replace the core of LPD with Ca phosphate (CaP) which dissolves at the acidic endosome pH. We expect that dissolved CaP will significantly increase the osmotic pressure of the endosome and induce swelling and rupture of the organelle, resulting in the release of the encapsulated siRNA. The proposed improvement of the non-viral vector will combine high level of tumor uptake and efficient endosome release of the siRNA cargo. The other aim of the project will deal with tumors with not-so-leaky neovasculature. We have successfully prepared small bioactive lipoplex (SBL) by mixing siRNA and a cationic lipid, DOTAP, in a fluorohydrocarbon solvent at an elevated temperature and pressure. The resulting nanoparticles were small (30-50 nm), and active in transfection. We will further modify the surface of SBL with PEGylation and ligand tethering. We expect that the new nanoparticle will deliver siRNA to tumor cells in which the neovasculature is not so leaky.