A Phenylboronate-Functionalized Polyion Complex Micelle for ATP-Triggered Release of siRNA
A Phenylboronate-Functionalized Polyion Complex Micelle for ATP-Triggered Release of siRNA
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DOI：
10.1002/anie.201203360
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发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Kataoka, Kazunori
中科院分区：
文献类型：
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作者：
Naito, Mitsuru;Ishii, Takehiko;Kataoka, Kazunori
基于小干扰RNA（siRNA）的治疗提供了一个有吸引力的临床选择，因为它能够以高度序列特异性的方式沉默基因。[1]一个关键的挑战在于开发一种递送系统，该系统有助于保护siRNA免受内源性RNA酶降解，同时允许受控的药代动力学。一种有前景的方法是聚离子复合物（PIC）胶束的制剂，其仅通过阴离子siRNA和阳离子聚合物之间的静电相互作用在水性环境中自发形成。[2]随着对应的阳离子聚合物，代表性的聚（乙二醇）为基础的嵌段共聚物的通用设计，许多创造性的PIC为基础的策略已经出现，其中一些已经显示出令人鼓舞的体外基因沉默能力。[3-5]然而，通常，这些基于PIC的载体在生理条件下具有不稳定性，主要是因为siRNA的相对短的链长，即20-25个核苷酸，这导致差的热力学稳定性。因此，基于PIC的载体的稳定化使得在到达细胞内靶位点时程序性去稳定化（以释放siRNA）一直是令人关注的。目前的努力集中在以下三种代表性方法中的一种或组合：siRNA与归巢聚合物的共价缀合，[3，6-8]引入疏水部分以增强核心聚集，[9-11]和通过二硫键桥接交联核心聚集体。[5，12]因此，这些方法的组合通常导致高度复杂的结构和制备方法。在这里，我们描述了一个复杂的解决方案，可以显着简化PIC的合成。它使用苯基硼酸酯官能团，其结合了所有上述稳定方法（方案1），同时保持对环境敏感性的宽控制窗口。苯基硼酸（PBA）是一种合成分子，能够与包括核糖环上的1，2-或1，3-顺式二醇形成可逆的共价酯，[13-15]核糖环上的结构存在于RNA和几种核糖核酸的3о末端。由于这种性质，PBA在历史上被用作亲和层析中RNA的配体。[16]因此，这种结合性质为siRNA与悬垂PBA基团的化学缀合提供了一种简便的途径。一旦静电凝聚到PIC中，平衡结合的机会增加，其中分子间交联也可以形成，因为在双链siRNA的30端的双二齿核糖排列，从而进一步稳定复合物。此外，PBA的独特之处在于，它的疏水性水平取决于酸解离的程度而发生戏剧性的逆转;[17]它在不带电荷时具有强疏水性，但当pH值高于其pKa时带负电荷时变得亲水。如图1所示，PBA和siRNA之间的结合基本上是可逆的平衡过程，取决于每种物质的浓度。这些特征可用于微调或切换复合物的稳定性，这与创建对细胞间和细胞内环境敏感的系统有关。本文中，我们证明了PBA辅助的PIC胶束可以被定制以显示出对核糖浓度变化的响应（这与细胞内环境中的事件是平行的）的显著破坏并伴随着siRNA的释放。首先制备了平台阳离子聚合物聚乙二醇-嵌段聚赖氨酸（PEG-b-PLys），定量测定了其赖氨酸残基，并将其与聚乙二醇-嵌段聚赖氨酸（PEG-b-PLys）进行了比较。结果表明，PBA-b-PLys具有良好的稳定性和稳定性。
Therapeutics based on small interfering RNA (siRNA) offer an attractive clinical option because of its ability to silence genes in a highly sequence-specific manner.[1] A key challenge lies in developing a delivery system that helps protect the siRNAs from endogenous RNase degradation while allowing for controlled pharmacokinetics. One promising approach is a formulation of polyion complex (PIC) micelles that spontaneously form in an aqueous environment simply through electrostatic interactions between the anionic siRNA and cationic polymers.[2] With versatile designs of the counterpart cationic polymers, representative poly (ethylene glycol) based block co-polymers, many creative PIC-based strategies have emerged, some of which have shown encouraging in vitro gene silencing abilities.[3–5] However, in general, these PIC-based carriers suffer from instability under physiological conditions, primarily because of the relatively short chain length of the siRNA, that is 20–25 nucleotides, which results in poor thermodynamic stability. Therefore, stabilization of the PIC-based carriers so that programmed destabilization upon arrival at the site of intracellular targets (to release siRNA) has been of interest. Current efforts have focused on either one or combinations of the following three representative approaches: covalent conjugation of siRNAs to a homing polymer,[3, 6–8] introduction of hydrophobic moieties to reinforce the core-aggregation,[9–11] and crosslinking the core aggregate by disulfide bridging.[5, 12] As such, the combination of these approaches often results in a highly complex structure and method of preparation. Herein, we describe a sophisticated solution that can remarkably simplify the synthesis of PICs. It uses a phenylboronate functionality, which incorporates all of the aforementioned methods of stabilization (Scheme 1) while maintaining a wide window of control for environmental sensitivity. Phenylboronic acid (PBA) is a synthetic molecule capable of forming reversible covalent esters with 1, 2-or 1, 3-cis-diols including on a ribose ring,[13–15] a structure which is present at the 3о end of RNAs and several kinds of ribonucleotides. Because of this property, PBA has historically been used as a ligand for RNA in affinity chromatography.[16] Therefore, this binding property offers a facile route for chemical conjugation of siRNAs to the pendant PBA groups. Once electrostatically condensed into the PIC, the chances of equilibrium binding are increased, in which intermolecular cross-links could also form because of the bis-bidentate ribose arrangement at the 3о end of the double-stranded siRNA, thereby further stabilizing the complex. Furthermore, PBA is unique in that it undergoes a dramatic inversion in its level of hydrophobicity depending on the degree of acid disassociation;[17] it is strongly hydrophobic when uncharged but it becomes hydrophilic when negatively charged at pH values above its pKa. As shown in Figure 1, the binding between PBA and siRNAs is essentially a reversible equilibrium process dependent on the concentrations of each species. These features can be used to fine-tune or switch the stability of the complex, which is relevant to creating a system that is sensitive to the inter-and intracellular environments. Herein, we demonstrate that the PBA-assisted PIC micelles can be tailored to exhibit a dramatic disruption accompanied by the release of siRNAs in response to a change in the ribose concentration (which parallels events in the intracellular environment).A platform cationic polymer poly (ethylene glycol)-blockpoly (l-lysine)(PEG-b-PLys) was first prepared, the lysine residues of which were quantitatively …