Tangsangasaksri Montira, Takemoto Hiroyasu, Naito Mitsuru, Maeda Yoshinori, Sueyoshi Daiki, Kim Hyun Jin, Miura Yutaka, Ahn Jooyeon, Azuma Ryota, Nishiyama Nobuhiro, Miyata Kanjiro, Kataoka Kazunori
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Polymer Chemistry Division, Chemical Resources Laboratory, Tokyo Institute of Technology , R1-11, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan.
Biomacromolecules. 2016 Jan 11;17(1):246-55. doi: 10.1021/acs.biomac.5b01334. Epub 2015 Dec 8.
Small interfering RNA (siRNA) needs an efficient delivery vehicle to reach the cytoplasm of target cells for successful RNA interference (RNAi) therapy. This study aimed to develop an siRNA-loaded polyion complex (PIC) micelle equipped with a smart polymeric shell featuring tumor targetability and endosome escapability for enhanced RNAi activity in cancer cells. To this end, an acidic pH-responsive polypeptide was designed to exert a stepwise change in its charged state from negative to modestly positive and highly positive in response to slightly acidic environment of tumor (pH ∼6.7) and further lowered-pH condition of late endosomal compartments (pH ∼5.0), respectively, for selective binding to cancer cell surface and subsequent endosome disruption. This polypeptide, termed PAsp(DET-CDM/DBCO), was synthesized by introducing acid-labile carboxydimethyl maleate (CDM) and dibenzylcyclooctyne (DBCO) moieties into a polyaspartamide derivative bearing two-repeated aminoethylene side chains (PAsp(DET)). Then, PAsp(DET-CDM/DBCO) was installed on the surface of disulfide cross-linked PIC micelles prepared from cholesterol-modified siRNA (Chol-siRNA) and azide-poly(ethylene glycol)-b-poly[(3-mercaptopropylamidine)-L-lysine] (N3-PEG-b-PLys(MPA)) through the copper-free click reaction. Successful PAsp(DET-CDM/DBCO) coverage of PIC micelles was confirmed by a significant decrease in ζ-potential as well as a narrowly distributed size of 40 nm. The PAsp(DET-CDM/DBCO)-installed micelles significantly improved the gene-silencing efficiency in cultured lung cancer cells, compared with nonmodified control micelles, especially after incubation at pH 6.7. This improved silencing activity was nicely correlated with the facilitated cellular uptake of siRNA payloads at the acidic pH and the efficient endosomal escape. These results demonstrate that the acidic pH-responsive polypeptide shell is a promising design strategy for tumor-targeted siRNA delivery.
小分子干扰RNA（siRNA）需要一种高效的递送载体才能进入靶细胞的细胞质，以成功实现RNA干扰（RNAi）治疗。本研究旨在开发一种负载siRNA的聚离子复合物（PIC）胶束，其具有智能聚合物外壳，具备肿瘤靶向性和内体逃逸能力，以增强癌细胞中的RNAi活性。为此，设计了一种酸性pH响应多肽，使其在肿瘤的微酸性环境（pH ∼6.7）和晚期内体区室的更低pH条件（pH ∼5.0）下，其电荷状态分别从负向适度正和高度正发生逐步变化，以便分别与癌细胞表面选择性结合并随后破坏内体。这种多肽称为PAsp(DET-CDM/DBCO)，是通过将酸不稳定的马来酸羧基二甲基酯（CDM）和二苄基环辛炔（DBCO）部分引入带有两个重复氨基乙撑侧链的聚天冬酰胺衍生物（PAsp(DET)）中合成的。然后，通过无铜点击反应将PAsp(DET-CDM/DBCO)安装在由胆固醇修饰的siRNA（Chol-siRNA）和叠氮基-聚（乙二醇）-b-聚[(3-巯基丙基脒)-L-赖氨酸]（N3-PEG-b-PLys(MPA)）制备的二硫键交联的PIC胶束表面。PIC胶束成功覆盖PAsp(DET-CDM/DBCO)通过ζ电位的显著降低以及40 nm的窄分布尺寸得到证实。与未修饰的对照胶束相比，安装了PAsp(DET-CDM/DBCO)的胶束显著提高了培养的肺癌细胞中的基因沉默效率，尤其是在pH 6.7孵育后。这种改善的沉默活性与在酸性pH下促进siRNA有效载荷的细胞摄取以及有效的内体逃逸密切相关。这些结果表明，酸性pH响应多肽外壳是一种有前景的肿瘤靶向siRNA递送设计策略。
