Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Biomaterials. 2012 Mar;33(9):2770-9. doi: 10.1016/j.biomaterials.2011.12.022. Epub 2011 Dec 24.
The delivery of siRNA therapeutics owes its success to the development of carrier systems with high efficacy and minimum toxicity. Here, cationic polyaspartamide derivatives with a regulated number and spacing of positively charged amino groups in the side chain were prepared from a single platform polymer of poly(β-benzyl l-aspartate) to assess their availability as siRNA carriers through polyion complex (PIC) formation. These polymers have 1,2-diaminoethane, 1,3-diaminopropane, and N,N'-bis(2-aminoethyl)-1,2-diaminoethane moieties in the side chain, and are termed as PAsp(DET), PAsp(DPT), and PAsp(TEP), respectively. siRNA-loaded PICs stable in serum-containing media were formed from PAsp(TEP) and PAsp(DPT) with two positive charges in the side chain at pH 7.4, whereas no such stable PIC was obtained from PAsp(DET) with only a single charge in the side chain, suggesting facilitated multivalent interactions with siRNA molecules to increase the PIC stability. The PAsp(DPT) and PAsp(TEP) PICs stable in the serum-containing media underwent an appreciably enhanced uptake into cultured cells through endocytosis, and subsequently exerted effective endosomal escape for the significant silencing of target gene expression. Notably, PAsp(TEP) PIC displayed negligible cytotoxicity in sharp contrast to the highly toxic feature of PAsp(DPT) PIC. This cytotoxicity is apparently correlated with the minimal damage to the cytoplasmic membrane of cells exposed to PAsp(TEP) at pH 7.4 evidenced from the fluorescent dye (YO-PRO-1) permeation assay. There was, in turn, a significant increase in YO-PRO-1 permeability at endosomal pH of 5.5 for PAsp(TEP)-exposed cells, indicating that PAsp(TEP) exerts membrane damage in a pH-selective manner, and eventually facilitates the translocation of siRNA-loaded PIC from the acidic endosomal compartment into the cytoplasm for effective gene silencing without any severe toxicity at physiological conditions. This acidic pH modulated enhancement in membrane damage of PAsp(TEP) may be explained by an increased protonation of the arrayed amino groups in the side chain that strongly perturb the endosomal membrane integrity. Eventually, PAsp(TEP) with a side chain array of pH-sensitive amino groups was demonstrated to be a promising component for constructing siRNA carriers exerting effective gene silencing in a less toxic context.
阳离子聚天冬酰胺衍生物的成功，归功于高效、低毒的载体系统的发展。在这里，我们从聚（β-苄基 L-天冬氨酸）的单一平台聚合物中制备了带有侧链中带正电荷的氨基数量和间距得到调控的聚天冬酰胺衍生物，以评估它们作为通过聚离子复合物（PIC）形成的 siRNA 载体的可用性。这些聚合物在侧链中具有 1,2-二氨基乙烷、1,3-二氨基丙烷和 N,N'-双（2-氨基乙基）-1,2-二氨基乙烷部分，分别称为 PAsp（DET）、PAsp（DPT）和 PAsp（TEP）。在 pH 7.4 时，带两个侧链正电荷的 PAsp（TEP）和 PAsp（DPT）可形成稳定负载 siRNA 的 PIC，而在 pH 7.4 时仅带有单个侧链电荷的 PAsp（DET）则不能形成稳定的 PIC，这表明与 siRNA 分子的多价相互作用促进了 PIC 的稳定性。在含有血清的培养基中稳定的 PAsp（DPT）和 PAsp（TEP）PIC 通过内吞作用显著增强了对培养细胞的摄取，随后有效促进了内涵体逃逸，从而显著抑制了靶基因的表达。值得注意的是，PAsp（TEP）PIC 在 pH 7.4 时对细胞质膜的损伤最小，与 PAsp（DPT）PIC 的高毒性特征形成鲜明对比。这种细胞毒性显然与暴露于 pH 7.4 的 PAsp（TEP）的细胞质膜的最小损伤有关，这从荧光染料（YO-PRO-1）渗透试验中得到证实。对于 PAsp（TEP）暴露的细胞，在内涵体 pH 为 5.5 时，YO-PRO-1 的渗透率显著增加，表明 PAsp（TEP）以 pH 选择性的方式发挥膜损伤作用，最终促进负载 siRNA 的 PIC 从酸性内涵体隔室转运到细胞质中，在生理条件下有效沉默基因而没有任何严重的毒性。这种酸性 pH 调节的 PAsp（TEP）膜损伤增强，可能是由于侧链中排列的氨基基团的质子化增加，强烈扰乱了内涵体膜的完整性。最终，具有 pH 敏感的氨基酸侧链排列的 PAsp（TEP）被证明是构建在毒性较低的情况下有效沉默基因的 siRNA 载体的有前途的成分。
