Department of Pharmacology, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA.
ACS Nano. 2011 Nov 22;5(11):9246-55. doi: 10.1021/nn203503h. Epub 2011 Nov 1.
The endosomal barrier is a major bottleneck for the effective intracellular delivery of siRNA by nonviral nanocarriers. Here, we report a novel amphotericin B (AmB)-loaded, dual pH-responsive micelleplex platform for siRNA delivery. Micelles were self-assembled from poly(2-(dimethylamino)ethyl methacrylate)-block-poly(2-(diisopropylamino)ethyl methacrylate) (PDMA-b-PDPA) diblock copolymers. At pH 7.4, AmB was loaded into the hydrophobic PDPA core, and siRNA was complexed with a positively charged PDMA shell to form the micelleplexes. After cellular uptake, the PDMA-b-PDPA/siRNA micelleplexes dissociated in early endosomes to release AmB. Live cell imaging studies demonstrated that released AmB significantly increased the ability of siRNA to overcome the endosomal barrier. Transfection studies showed that AmB-loaded micelleplexes resulted in significant increase in luciferase (Luc) knockdown efficiency over the AmB-free control. The enhanced Luc knockdown efficiency was abolished by bafilomycin A1, a vacuolar ATPase inhibitor that inhibits the acidification of the endocytic organelles. These data support the central hypothesis that membrane poration by AmB and increased endosomal swelling and membrane tension by a "proton sponge" polymer provided a synergistic strategy to disrupt endosomes for improved intracellular delivery of siRNA.
内涵体屏障是通过非病毒纳米载体有效实现 siRNA 细胞内递呈的主要瓶颈。在此，我们报告了一种新型两性霉素 B（AmB）负载的、双重 pH 响应性胶束复合物平台，用于 siRNA 递呈。胶束由聚[2-（二甲氨基）乙基甲基丙烯酸酯]-嵌段-聚[2-（二异丙基氨基）乙基甲基丙烯酸酯]（PDMA-b-PDPA）两亲性嵌段共聚物自组装而成。在 pH 7.4 时，AmB 被载入疏水性 PDPA 内核，siRNA 与带正电荷的 PDMA 壳复合形成胶束复合物。细胞摄取后，PDMA-b-PDPA/siRNA 胶束复合物在早期内涵体中解离，释放 AmB。活细胞成像研究表明，释放的 AmB 显著提高了 siRNA 克服内涵体屏障的能力。转染研究表明，负载 AmB 的胶束复合物导致荧光素酶（Luc）的敲低效率显著高于无 AmB 对照。这种增强的 Luc 敲低效率被溶酶体 ATP 酶抑制剂巴弗洛霉素 A1 所消除，溶酶体 ATP 酶抑制剂可以抑制内吞细胞器的酸化。这些数据支持了一个中心假说，即 AmB 的膜穿孔作用和“质子海绵”聚合物引起的内涵体肿胀和膜张力增加提供了一种协同策略，以破坏内涵体，从而提高 siRNA 的细胞内递呈效率。
