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

 描述（由申请人提供）：多药耐药（MDR）肿瘤的有效治疗仍然是癌症治疗中尚未解决的重要问题。MDR细胞中过表达的某些蛋白质，如P-糖蛋白（Pgp）或生存素，参与了这些细胞对传统化疗的耐药性。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。这种方法与传统化疗的结合可能为MDR肿瘤提供一种有前途的策略。 siRNA的临床应用受到其稳定性和递送问题的限制。我们最近提出了一种用于siRNA的聚（乙二醇）-磷脂酰乙醇胺（PEG-PE）修饰的G（4）-PAMAM纳米载体。我们还从这种新的G（4）- PAMAM-PEG-PE和PEG-PE构建体开发了一种独特的基于树枝状聚合物的混合胶束系统。这种胶束可以通过树枝状聚合物部分复合siRNA，并将难溶性抗癌药物捕获在其脂质核心内。这样的系统具有高胶束化效率，具有低CMC和较高的药物负载;稳定和保护siRNA免受酶促降解;由于其正电荷而提供增强的细胞结合、渗透和内体逃逸;并且由于PEG化而显示出较小的细胞毒性。它将允许siRNA和药物在一个纳米制剂中的共递送，如果基于树枝状聚合物的胶束用靶向配体进行表面修饰，则包括靶向的纳米制剂。 我们的一般假设是PEG-脂质修饰的基于树枝状聚合物的聚合物混合胶束将通过树枝状聚合物部分复合siRNA并将难溶性抗癌药物加载到核心中，从而产生siRNA/药物共加载的纳米制剂，其可以通过表面附着的抗癌抗体（我们早期开发的mAb 2C 5）另外靶向肿瘤，并将用作对抗MDR肿瘤的有效手段。本研究的具体目的如下：（1）制备、表征和评价PAMAM-PEG-PE/siRNA（P-gp和Survivin siRNA）复合物及其与PEG-PE和抗癌药物的混合胶束纳米制剂（多柔比星或紫杉醇）修饰或未修饰的mAb 2C 5;（2）使用非靶向的、单克隆抗体靶向的siRNA/共载药的树状大分子纳米制剂，研究Aim 1的非靶向或单克隆抗体靶向的siRNA/共载药的树状大分子纳米制剂与多种MDR癌细胞的体外相互作用。MDR细胞作为对照：（a）跟踪siRNA和药物的细胞缔合和细胞内积累;（B）研究纳米制剂在MDR细胞中下调靶基因的效率;（c）使用乱序siRNA作为对照，评价它们在MDR细胞中的细胞毒性和MDR逆转;（3）为了证明有效的siRNA/药物共转染的可能性，在两种不同的体内模型中使用开发的系统和根据目标2选择的治疗剂递送至MDR肿瘤和增强肿瘤治疗：（a）研究选择的非靶向和靶向siRNA/共负载药物的纳米制剂在荷瘤小鼠中的肿瘤积累;（B）使用乱序siRNA作为对照来评价它们在肿瘤生长抑制中的活性。

 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.