Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
Bioconjug Chem. 2013 Mar 20;24(3):398-407. doi: 10.1021/bc300486k. Epub 2013 Feb 22.
RNA interference (RNAi) drugs have significant therapeutic potential, but delivery systems with appropriate efficacy and toxicity profiles are still needed. Here, we describe a neutral, ampholytic polymeric delivery system based on conjugatable diblock polymer micelles. The diblock copolymer contains a hydrophilic poly[N-(2-hydroxypropyl)methacrylamide-co-N-(2-(pyridin-2-yldisulfanyl)ethyl)methacrylamide) (poly[HPMA-co-PDSMA]) segment to promote aqueous stability and facilitate thiol-disulfide exchange reactions and a second ampholytic block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The poly[(HPMA-co-PDSMA)-b-(PAA-co-DMAEMA-co-BMA)] was synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization with an overall molecular weight of 22 000 g/mol and a PDI of 1.88. Dynamic light scattering and fluorescence measurements indicated that the diblock copolymers self-assemble under aqueous conditions to form polymeric micelles with a hydrodynamic radius and critical micelle concentration of 25 nm and 25 μg/mL, respectively. Red blood cell hemolysis experiments show that the neutral hydrophilic micelles have potent membrane destabilizing activity at endosomal pH values. Thiolated siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was directly conjugated to the polymeric micelles via thiol exchange reactions with the pyridal disulfide groups present in the micelle corona. Maximum silencing activity in HeLa cells was observed at a 1:10 molar ratio of siRNA to polymer following a 48 h incubation period. Under these conditions 90% mRNA knockdown and 65% protein knockdown at 48 h was achieved with negligible toxicity. In contrast the polymeric micelles lacking a pH-responsive endosomalytic segment demonstrated negligible mRNA and protein knockdown under these conditions. The potent mRNA knockdown and excellent biocompatibility of the neutral siRNA conjugates demonstrate the potential utility of this carrier design for delivering therapeutic siRNA drugs.
RNA 干扰 (RNAi) 药物具有显著的治疗潜力，但仍需要具有适当疗效和毒性特征的递送系统。在这里，我们描述了一种基于可共轭两亲嵌段聚合物胶束的中性、两性聚合物递送系统。该两亲嵌段共聚物包含亲水性聚N-(2-羟丙基)甲基丙烯酰胺-co-N-(2-(吡啶-2-基二硫基)乙基)甲基丙烯酰胺 段，以促进水稳定性并促进巯基-二硫键交换反应和由丙基丙烯酸 (PAA)、二甲基氨基乙基甲基丙烯酸酯 (DMAEMA) 和丁基甲基丙烯酸酯 (BMA) 组成的第二两性嵌段。聚[(HPMA-co-PDSMA)-b-(PAA-co-DMAEMA-co-BMA)] 是使用可逆加成-断裂链转移 (RAFT) 聚合合成的，其分子量为 22000g/mol，PDI 为 1.88。动态光散射和荧光测量表明，两亲嵌段共聚物在水相条件下自组装形成聚合物胶束，其水动力半径和临界胶束浓度分别为 25nm 和 25μg/mL。红细胞溶血实验表明，中性亲水胶束在内涵体 pH 值下具有很强的膜破坏活性。通过与胶束冠层中存在的吡啶二硫基团的巯基交换反应，将靶向甘油醛 3-磷酸脱氢酶 (GAPDH) 的硫代化 siRNA 直接共轭到聚合物胶束上。在孵育 48 小时后，siRNA 与聚合物的摩尔比为 1:10 时，在 HeLa 细胞中观察到最大的沉默活性。在这些条件下，48 小时时实现了 90%的 mRNA 敲低和 65%的蛋白质敲低，且毒性可忽略不计。相比之下，在这些条件下，缺乏 pH 响应内涵体片段的聚合物胶束几乎没有实现 mRNA 和蛋白质的敲低。中性 siRNA 缀合物的强大的 mRNA 敲低和优异的生物相容性证明了这种载体设计用于递送治疗性 siRNA 药物的潜力。
