Novel nanoparticles for siRNA delivery

描述（由申请人提供）：近年来，我们已经成功地修饰了脂质体/鱼精蛋白/DNA（LPD）纳米颗粒制剂，用于在异种移植模型中将siRNA靶向递送至人肺癌细胞。这种核/壳自组装纳米颗粒含有由两个阳离子脂质双层包裹的紧凑的鱼精蛋白/DNA/siRNA核。当纳米颗粒与聚乙二醇（PEG）-磷脂缀合物（DSPE-PEG）一起孵育时，外层双层被剥离，但内层双层以高度的PEG化存活下来。密集堆积的表面PEG形成刷保护层以屏蔽纳米颗粒的阳离子电荷并减少血清蛋白的调理作用。结果是肝脏和脾脏的摄取程度非常低，而肿瘤摄取水平非常高，高达60-80%注射剂量/g组织。如果靶向配体茴香酰胺被拴在PEG的远端，靶向纳米颗粒有效地递送siRNA以沉默整个肿瘤中的靶基因。然而，在更仔细地观察递送的siRNA时，大部分剂量被隔离在内体中并且不是生物可利用的。这是因为该制剂不具有任何内体裂解活性。因此，我们建议用磷酸钙（CaP）取代LPD的核心，磷酸钙（CaP）在酸性内体pH下溶解。我们预计溶解的CaP将显著增加内体的渗透压，并诱导细胞器的肿胀和破裂，从而释放封装的siRNA。所提出的非病毒载体的改进将联合收割机结合高水平的肿瘤摄取和siRNA货物的有效内体释放。该项目的另一个目的是处理具有不那么渗漏的新血管的肿瘤。我们已经成功地制备了小的生物活性lipoplex（SBL）的混合siRNA和阳离子脂质，DOTAP，在氟代烃溶剂在升高的温度和压力。所得纳米颗粒较小（30-50 nm），并且在转染中具有活性。我们将进一步修饰SBL的表面与聚乙二醇化和配体拴系。我们希望这种新的纳米颗粒能够将siRNA递送到新血管不那么渗漏的肿瘤细胞中。

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.