Department of Bioengineering, University of Washington, Seattle Washington 98195, USA.
Mol Pharm. 2010 Apr 5;7(2):442-55. doi: 10.1021/mp9002255.
Small interfering RNA (siRNA)-based therapies have great potential for the treatment of debilitating diseases such as cancer, but an effective delivery strategy for siRNA is elusive. Here, pH-responsive complexes were developed for the delivery of siRNA in order to sensitize drug-resistant ovarian cancer cells (NCI/ADR-RES) to doxorubicin. The electrostatic complexes consisted of a cationic micelle used as a nucleating core, siRNA, and a pH-responsive endosomolytic polymer. Cationic micelles were formed from diblock copolymers of dimethylaminoethyl methacrylate (pDMAEMA) and butyl methacrylate (pDbB). The hydrophobic butyl core mediated micelle formation while the positively charged pDMAEMA corona enabled siRNA condensation. To enhance cytosolic delivery through endosomal release, a pH-responsive copolymer of poly(styrene-alt-maleic anhydride) (pSMA) was electrostatically complexed with the positively charged siRNA/micelle to form a ternary complex. Complexes exhibited size (30-105 nm) and charge (slightly positive) properties important for endocytosis and were found to be noncytotoxic and mediate uptake in >70% of ovarian cancer cells after 1 h of incubation. The pH-responsive ternary complexes were used to deliver siRNA against polo-like kinase 1 (plk1), a gene upregulated in many cancers and responsible for cell cycle progression, to ovarian cancer cell lines. Treatment resulted in approximately 50% reduction of plk1 gene expression in the drug-resistant NCI/ADR-RES ovarian cancer cell model and in the drug-sensitive parental cell line, OVCAR8. This knockdown functionally sensitized NCI/ADR-RES cells to doxorubicin at levels similar to OVCAR8. Sensitization occurred through a p53 signaling pathway, as indicated by caspase 3/7 upregulation following plk1 knockdown and doxorubicin treatment, and this effect could be abrogated using a p53 inhibitor. To demonstrate the potential for dual delivery from this polymer system, micelle cores were subsequently loaded with doxorubicin and utilized in ternary complexes to achieve cell sensitization through simultaneous siRNA and drug delivery from a single carrier. These results show knockdown of plk1 results in sensitization of multidrug resistant cells to doxorubicin, and this combination of gene silencing and small molecule drug delivery may prove useful to achieve potent therapeutic effects.
基于小干扰 RNA（siRNA）的疗法在治疗癌症等衰弱性疾病方面具有巨大潜力，但有效的 siRNA 递药策略仍难以捉摸。本研究开发了 pH 响应性复合物，用于递送 siRNA，以增加耐多柔比星的卵巢癌细胞（NCI/ADR-RES）对多柔比星的敏感性。这些静电复合物由用作成核核心的阳离子胶束、siRNA 和 pH 响应性的内涵体溶酶体聚合物组成。阳离子胶束由甲基丙烯酰氧乙基二甲胺基（pDMAEMA）和正丁基甲基丙烯酸酯（pDbB）的嵌段共聚物形成。疏水性正丁基核心介导胶束形成，而带正电荷的 pDMAEMA 冠使 siRNA 发生凝聚。为了通过内涵体释放增强细胞质内递送，将聚（苯乙烯-alt-马来酸酐）（pSMA）的 pH 响应性共聚物静电复合到带正电荷的 siRNA/胶束上，形成三元复合物。复合物具有 30-105nm 的大小（粒径）和带轻微正电荷的特性，这对于内吞作用很重要，并且在孵育 1 小时后，复合物被发现对卵巢癌细胞的摄取率超过 70%，且无细胞毒性。用 pH 响应性三元复合物递送至多柔比星耐药的 NCI/ADR-RES 卵巢癌细胞模型和药物敏感的亲本细胞系 OVCAR8 中的 polo 样激酶 1（plk1）的 siRNA。治疗导致耐药的 NCI/ADR-RES 卵巢癌细胞模型和药物敏感的亲本细胞系 OVCAR8 中 plk1 基因表达减少约 50%。这种敲低通过 caspase 3/7 的上调使 NCI/ADR-RES 细胞对多柔比星的敏感性与 OVCAR8 相似，从而使细胞功能敏感化。这种作用可通过 p53 抑制剂阻断。为了证明该聚合物系统的双重递药潜力，随后将胶束核心加载多柔比星，并将其用于三元复合物中，通过从单个载体同时递送 siRNA 和药物来实现细胞敏感化。这些结果表明，plk1 的敲低导致多药耐药细胞对多柔比星的敏感性增加，这种基因沉默和小分子药物递药的组合可能有助于实现有效的治疗效果。
