Novel nanoparticles for siRNA delivery

描述(申请人提供)：近年来，我们成功地改进了脂质体/鱼精蛋白/DNA(LPD)纳米颗粒配方，用于在异种移植模型中将siRNA靶向输送到人肺癌细胞。这种核/壳自组装纳米颗粒包含一个紧密的鱼精蛋白/DNA/siRNA核心，它被两个阳离子脂质双层包裹着。当纳米粒子与聚乙二醇磷脂偶联物(DSPE-PEG)孵育时，外层双层被剥离，但内双层保留下来，聚乙二醇化程度很高。密实堆积的聚乙二醇表面形成了一层刷状保护层，屏蔽了纳米粒子的阳离子电荷，并减少了血清蛋白的调理作用。结果是肝脏和脾的摄取程度非常低，而肿瘤的摄取水平非常高，每克组织高达60%-80%的注射剂量。如果靶向配体苯甲酰胺被拴在聚乙二醇远端，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，仔细观察传递的siRNA，大部分剂量被隔离在内吞体内，无法在生物上利用。这是因为该制剂不具有任何内体裂解活性。因此，我们建议用在酸性内涵体pH下溶解的磷酸钙(CAP)来取代LPD的核心。我们预计，溶解的帽将显著增加内体的渗透压，并导致细胞器肿胀和破裂，导致包裹的siRNA释放。拟议的非病毒载体的改进将结合高水平的肿瘤摄取和高效的siRNA货物的内体释放。该项目的另一个目标是处理新生血管不那么渗漏的肿瘤。我们通过将siRNA和阳离子脂类DOTAP混合在氟烃溶剂中，在高温和压力下成功地制备了具有生物活性的小分子脂复合物(SBL)。得到的纳米颗粒很小(30-50 nm)，并且在转染过程中具有活性。我们将进一步对SBL表面进行聚乙二醇化和配体系链修饰。我们预计这种新的纳米颗粒将把siRNA运送到肿瘤细胞中，而肿瘤细胞中的新生血管不会如此渗漏。 与公共卫生相关：该项目将解决向肿瘤传递siRNA方面的两个紧迫问题。第一个是提高siRNA从内体到细胞质的释放。另一种是将siRNA运送到血管系统不那么漏水的肿瘤中。如果成功，该项目将极大地推动siRNA作为癌症治疗药物。

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. PUBLIC HEALTH RELEVANCE: The project will address two pressing issues in delivering siRNA to the tumor. The first is to improve the release of siRNA from the endosome to the cytoplasm. The other is to transport siRNA to tumors with not-so-leaky vasculature. If successful, the project will significantly advance siRNA as cancer therapeutics.