Segura Tatiana, Hubbell Jeffrey A
Ecole Polytechnique Fédérale de Lausanne, Institute of Bioengineering and Institute of Chemical Sciences and Engineering, CH-1015, Lausanne, Switzerland.
Bioconjug Chem. 2007 May-Jun;18(3):736-45. doi: 10.1021/bc060284y. Epub 2007 Mar 15.
The ability to specifically down-regulate gene expression using the RNAi pathway in mammalian cells has tremendous potential in therapy and in basic science. However, delivery systems capable of efficient and biocompatible delivery of siRNA to target cells are not yet satisfactory. Here, we report the synthesis and in vitro characterization of ABC triblock copolymers that self-assemble with siRNA based on electrostatics and with each other by hydrophobic interactions. The ABC triblock copolymer is based on poly(ethylene glycol) (PEG), poly(propylene sulfide) (PPS), and a positively charged peptide (PEG-PPS-peptide). The diblock copolymer PEG(45)-PPS(5,10) was synthesized using anionic polymerization of propylene sulfide upon a PEG macroinitiator, and the peptide domain was coupled to the PPS terminus using a disulfide exchange reaction with an N-terminal cysteine residue on the peptide. The peptides were designed to interact electrostatically with siRNA, selecting the TAT peptide domain of HIV (RKKRRQRRR) and an oligolysine (Lys(9)). The resulting triblock copolymers were able to self-assemble with siRNA as demonstrated by dynamic light scattering and gel electrophoresis. Complex size was found to be dependent on the amount of polymer used (charge ratio) and the length of the hydrophobic PPS block, achieving sizes ranging from 171 nm to 601 nm. Cell internalization and gene expression down-regulation studies showed that the triblock copolymers are able to transport siRNA inside the cell and mediate gene expression down-regulation, with the amount of internalization and gene transfer affected by charge ratio, PPS length, and the presence of serum. The proposed triblock was able to mediate gene expression down-regulation of GAPDH, achieving up to 90.5% +/- 0.02% down-regulation.
在哺乳动物细胞中利用RNAi途径特异性下调基因表达的能力在治疗和基础科学领域具有巨大潜力。然而，能够将siRNA高效且生物相容地递送至靶细胞的递送系统仍不尽人意。在此，我们报告了ABC三嵌段共聚物的合成及其体外特性，该共聚物基于静电作用与siRNA自组装，并通过疏水相互作用彼此组装。ABC三嵌段共聚物基于聚乙二醇（PEG）、聚硫化丙烯（PPS）和带正电荷的肽（PEG-PPS-肽）。二嵌段共聚物PEG(45)-PPS(5,10)通过在PEG大分子引发剂上进行硫化丙烯的阴离子聚合反应合成，肽结构域通过与肽上N端半胱氨酸残基的二硫键交换反应与PPS末端偶联。这些肽被设计用于与siRNA进行静电相互作用，选用了HIV的TAT肽结构域（RKKRRQRRR）和一个寡聚赖氨酸（Lys(9)）。动态光散射和凝胶电泳表明，所得的三嵌段共聚物能够与siRNA自组装。发现复合物大小取决于所用聚合物的量（电荷比）和疏水PPS嵌段的长度，大小范围为171nm至601nm。细胞内化和基因表达下调研究表明，三嵌段共聚物能够将siRNA转运到细胞内并介导基因表达下调，内化量和基因转移受电荷比、PPS长度和血清存在的影响。所提出的三嵌段能够介导甘油醛-3-磷酸脱氢酶（GAPDH）的基因表达下调，下调幅度高达90.5%±0.02%。
