Lipid-dendrimer micellar nanocarriers for siRNA/drug co-delivery in MDR cancer

 描述(申请人提供)：多药耐药(MDR)肿瘤的有效治疗仍然是癌症治疗中一个重要的尚未解决的问题。在MDR细胞中过度表达的某些蛋白，如P-糖蛋白(Pgp)或Survivin，参与了这些细胞对传统化疗的耐药。通过应用相应的siRNA，可以实现癌细胞中此类蛋白质的特异性下调。这种方法与传统的化疗方法相结合，可能为治疗多药耐药肿瘤提供一种有前途的策略。SiRNA的稳定性和递送问题限制了其临床应用。我们最近提出了一种聚乙二醇磷脂酰乙醇胺(PEGPE)修饰的G(4)-PAMAM纳米siRNA载体。我们还用这种新型的G(4)-PAMAM-PEGPE和PEGPE构建了一种独特的树枝状大分子混合胶束体系。这种胶束可以通过树枝状大分子部分使siRNA复杂化，并将难溶的抗癌药物包裹在其脂核中。这种体系具有高胶束化效率和低CMC和较高的载药量；稳定和保护siRNA免受酶降解；由于其正电荷提供更强的细胞结合、穿透和内体逃逸；由于聚乙二醇化而表现出较低的细胞毒性。它将允许在一个纳米修复中同时传递siRNA和药物，包括如果树枝状大分子胶束用靶向配体进行表面修饰的话。我们的一般假设是，聚乙二醇脂修饰的树枝状大分子聚合物混合胶束将siRNA通过树枝状大分子部分络合，并将难于溶解的抗癌药物负载到核心，形成siRNA/药物共负载纳米修复，该纳米修复可通过我们先前开发的表面附着的抗癌抗体(MAb2C5)额外靶向肿瘤，将成为治疗多药耐药肿瘤的有效手段。(1)制备、表征和评价PAMAM-PEG-PE/siRNA(P-gp和Survivin siRNAs)复合物及其与mAb2C5修饰或未修饰的抗癌药物(阿霉素或紫杉醇)与聚乙二醇-PE的混合胶束纳米修复；(2)以非MDR细胞为对照，研究非靶向或单抗靶向的siRNA/药物共负载的树突状大分子纳米修复与多种MDR癌细胞的相互作用：(A)跟踪siRNA和药物在细胞内的结合和聚集；(B)研究纳米修复材料在多药耐药细胞中下调靶基因的效率；(C)以干扰siRNA为对照，评估其细胞毒作用和多药耐药逆转作用；(3)通过两种不同的体内模型和之后选择的治疗药物，验证所开发的系统有效地将siRNA/药物共输送到多药耐药肿瘤和增强肿瘤治疗的可能性；(A)研究选定的非靶向和靶向siRNA/药物共负载纳米修复材料在荷瘤小鼠体内的肿瘤蓄积；(B)以基因重组siRNA为对照，评估其抑制肿瘤生长的活性。

 DESCRIPTION (provided by applicant): The effective therapy of multidrug resistant (MDR) tumors remains an important unsolved problem in cancer therapy. Certain proteins over-expressed in MDR cells, such as P-glycoprotein (Pgp) or survivin, are involved in the resistance of these cells towards traditional chemotherapy. Specific down-regulation of such proteins in cancer cells can be achieved by applying the corresponding siRNA. The combination of this approach and traditional chemotherapy could provide a promising strategy against MDR tumors. The clinical use of siRNA is limited by problems with its stabilization and delivery. We have recently suggested a poly(ethylene glycol)-phosphatidyl ethanolamine (PEG-PE)-modified G(4)-PAMAM nanocarrier for siRNA. We have also developed a unique dendrimer-based mixed micellar system from this new G(4)- PAMAM-PEG-PE and PEG-PE constructs. Such micelles can complex siRNA via the dendrimer part and entrap poorly soluble anti-cancer drugs within their lipid core. Such a system has high micellization efficiency with low CMC and higher drug loading; stabilizes and protects siRNA against enzymatic degradation; provides enhanced cell binding, penetration, and endosomal escape because of its positive charge; and shows less cytotoxicity due to PEGylation. It will allow a co-delivery of siRNA and drug in one nanopreparation, including the targeted one if the dendrimer-based micelles are surface-modified with the targeting ligand. Our general hypothesis is that PEG-lipid-modified dendrimer-based polymeric mixed micelles will complex siRNA via the dendrimer part and load poorly soluble anticancer drugs into the core resulting in siRNA/drug-co-loaded nanopreparations, which can be additionally targeted to tumor by surface- attached anti-cancer antibodies (mAb 2C5 developed by us earlier), and will serve as an effective mean against MDR tumors. The following specific aims will be pursued: (1) To prepare, characterize, and evaluate PAMAM-PEG-PE/siRNA (P-gp and survivin siRNAs) complexes and their mixed micellar nanopreparations with PEG-PE and anti-cancer drugs (doxorubicin or paclitaxel) modified or non-modified with mAb 2C5; (2) To study the interaction of non-targeted or Ab-targeted siRNA/drug-co-loaded dendrimer-based nanopreparations from the Aim 1 with various MDR cancer cells in vitro using non-MDR cells as controls: (a) To follow cell association and intracellular accumulation of siRNA and drugs; (b) Investigate the efficiency of the nanopreparations in down-regulation of target genes in MDR cells; (c) Evaluate their cytotoxicity and MDR reversal in MDR cells using scrambled siRNA as control; (3) To demonstrate the possibility of efficient siRNA/drug co-delivery to MDR tumors and enhanced tumor therapy using the developed systems in two different in vivo models and treatment agents selected after the Aim 2: (a) To investigate tumor accumulation of chosen non-targeted and targeted siRNA/drug-co-loaded nanopreparations in tumor-bearing mice; (b) To evaluate their activity in tumor growth inhibition using scrambled siRNA as a control.