Suo Aili, Qian Junmin, Xu Minghui, Xu Weijun, Zhang Yaping, Yao Yu
Department of Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China.
State Key Laboratory for Mechanical Behavior of Materials, Xi'an Jiaotong University, Xi'an 710049, China.
Mater Sci Eng C Mater Biol Appl. 2017 Jul 1;76:659-672. doi: 10.1016/j.msec.2017.03.124. Epub 2017 Mar 18.
Co-delivery of chemotherapeutic drug and small interfering RNA (siRNA) within a single nanovehicle has emerged as a promising combination therapy approach to treating cancers because of their synergistic effect. Nanocarrier delivery systems with low cytotoxicity and high efficiency are needed for such a purpose. In this study, a novel folate-conjugated PEGylated cationic triblock copolymer, poly(acrylhydrazine)-block-poly(3-dimethylaminopropyl methacrylamide)-block-poly(acrylhydrazine) (PAH-b-PDMAPMA-b-PAH), was synthesized and evaluated as a stimuli-sensitive vehicle for the targeted co-delivery of doxorubicin (DOX) and Bcl-2 siRNA into breast cancer MCF-7 cells. The synthetic process of the PEGylated triblock copolymer involved sequential reversible addition-fragmentation chain transfer polymerization, PEGylation and removal of tert-butoxy carbamate protecting groups. Folate-conjugated and/or -unconjugated poly(ethylene glycol) segments were grafted onto PAH-b-PDMAPMA-b-PAH via a reduction-sensitive disulfide linkage. The synthetic polymers were characterized by H NMR and gel permeation chromatography. The PEGylated triblock copolymer could chemically conjugate DOX onto PAH blocks via pH-responsive hydrazone bonds and simultaneously complex negatively charged Bcl-2 siRNA with cationic PDMAPMA blocks through electrostatic interactions at N/P ratios≥32:1 to form multifunctional nanomicelleplexes. The nanomicelleplexes exhibited spherical shape, possessed a positively charged surface with a zeta potential of +22.5mV and had a desirable and uniform particle size of 187nm. In vitro release studies revealed that the nanomicelleplexes could release DOX and Bcl-2 siRNA in a reduction and pH dual-sensitive manner and the payload release was significantly enhanced in a reductive acidic environment mimicking the endosomes/lysosomes of cancer cells compared to under physiology conditions. Furthermore, the release of both DOX and siRNA was found to follow Higuchi kinetic model. Confocal laser scanning microscopy, flow cytometry and MTT analyses confirmed that, compared with folate-undecorated nanomicelleplexes, folate-decorated nanomicelleplexes could more effectively co-deliver DOX and Bcl-2 siRNA into MCF-7 cells and showed a stronger cell-killing effect. The pristine PEGylated triblock copolymer exhibited good cytocompatibility. Moreover, co-delivery of DOX and Bcl-2 siRNA achieved a significant synergistic antitumor efficacy. These findings suggested that the folate-decorated PEGylated cationic triblock copolymer might be a promising vehicle for targeted intracellular co-delivery of DOX and siRNA in MCF-7 cells, representing a potential clinical combination therapy for breast cancer treatment.
在单一纳米载体中共同递送化疗药物和小干扰RNA（siRNA），由于其协同效应，已成为一种有前景的癌症联合治疗方法。为此需要低细胞毒性和高效率的纳米载体递送系统。在本研究中，合成了一种新型叶酸共轭聚乙二醇化阳离子三嵌段共聚物，聚（丙烯酸肼）-嵌段-聚（3-二甲基氨基丙基甲基丙烯酰胺）-嵌段-聚（丙烯酸肼）（PAH-b-PDMAPMA-b-PAH），并评估其作为一种刺激敏感型载体，用于将阿霉素（DOX）和Bcl-2 siRNA靶向共同递送至乳腺癌MCF-7细胞中。聚乙二醇化三嵌段共聚物的合成过程包括顺序可逆加成-断裂链转移聚合、聚乙二醇化以及叔丁氧羰基保护基团的去除。叶酸共轭和/或未共轭的聚乙二醇链段通过还原敏感的二硫键接枝到PAH-b-PDMAPMA-b-PAH上。通过核磁共振氢谱（1H NMR）和凝胶渗透色谱对合成的聚合物进行了表征。聚乙二醇化三嵌段共聚物可通过pH响应性腙键将DOX化学偶联到PAH嵌段上，同时在N/P比≥32:1时通过静电相互作用使带负电荷的Bcl-2 siRNA与阳离子PDMAPMA嵌段复合，形成多功能纳米胶束复合物。纳米胶束复合物呈球形，表面带正电，zeta电位为+22.5mV，粒径为187nm，理想且均匀。体外释放研究表明，纳米胶束复合物能够以还原和pH双重敏感的方式释放DOX和Bcl-2 siRNA，与生理条件相比，在模拟癌细胞内体/溶酶体的还原酸性环境中，载药量释放显著增强。此外，发现DOX和siRNA的释放均符合Higuchi动力学模型。共聚焦激光扫描显微镜、流式细胞术和MTT分析证实，与未修饰叶酸的纳米胶束复合物相比，修饰叶酸的纳米胶束复合物能够更有效地将DOX和Bcl-2 siRNA共同递送至MCF-7细胞中，并表现出更强的细胞杀伤作用。原始的聚乙二醇化三嵌段共聚物表现出良好的细胞相容性。此外，DOX和Bcl-2 siRNA的共同递送实现了显著的协同抗肿瘤效果。这些发现表明，修饰叶酸的聚乙二醇化阳离子三嵌段共聚物可能是一种有前景的载体，用于在MCF-7细胞中靶向细胞内共同递送DOX和siRNA，代表了一种潜在的乳腺癌临床联合治疗方法。
