Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University , Suzhou 215123, China.
Department of General Surgery, Huashan Hospital, Fudan University , Shanghai 200040, China.
ACS Appl Mater Interfaces. 2017 Jul 19;9(28):23586-23601. doi: 10.1021/acsami.7b08534. Epub 2017 Jul 10.
The development of potent yet nontoxic membrane-penetrating materials is in high demand for effective intracellular gene delivery. We have recently developed α-helical polypeptides which afford potent membrane activities to facilitate intracellular DNA delivery via both endocytosis and the nonendocytic "pore formation" mechanism. Endocytosis will cause endosomal entrapment of the DNA cargo, while excessive "pore formation" would cause appreciable cytotoxicity. Additionally, helical polypeptides with stiff, rodlike structure suffer from low siRNA binding affinity. To address such critical issues, we herein incorporated various aromatic domains (benzyl, naphthyl, biphenyl, anthryl, and pyrenyl) into the side-chain terminals of guanidine-rich, helical polypeptides, wherein the flat-rigid shape, π-electronic structures of aromatic motifs "self-activated" the membrane-penetrating capabilities of polypeptides to promote intracellular gene delivery. Benzyl (Bn)- and naphthyl (Naph)-modified polypeptides demonstrated the highest DNA uptake level that outperformed the unmodified polypeptide, P2, by ∼4 fold. More importantly, compared with P2, Bn- and Naph-modified polypeptides allowed more DNA cargos to be internalized via the nonendocytic pathway, which significantly bypassed the endosomal entrapment and accordingly enhanced the transfection efficiency by up to 42 fold, outperforming PEI 25k as the commercial reagent by 3-4 orders of magnitude. The aromatic modification also improved the siRNA condensation capability of polypeptides, achieving notably enhanced gene-silencing efficiency against tumor necrosis factor-α to treat acute hepatic inflammation. Furthermore, we revealed that aromaticity-augmented membrane activity was accompanied by comparable or even significantly reduced "pore formation" capability, thus leading to diminished cytotoxicity at high concentrations. This study therefore provides a promising approach to manipulate the membrane activities and penetration mechanisms of polycations, which overcomes the multiple critical barriers preventing effective and safe gene delivery.
高效无毒的膜穿透材料的开发对于有效的细胞内基因传递非常重要。我们最近开发了α-螺旋多肽，它们具有强大的膜活性，通过内吞作用和非内吞作用的“孔形成”机制促进细胞内 DNA 传递。内吞作用会导致 DNA 货物被内体捕获，而过量的“孔形成”会导致明显的细胞毒性。此外，具有刚性棒状结构的螺旋多肽与 siRNA 的结合亲和力较低。为了解决这些关键问题，我们将各种芳族结构域（苄基、萘基、联苯基、蒽基和芘基）整合到胍基丰富的螺旋多肽的侧链末端，其中芳族结构的平面刚性形状和π电子结构“自激活”了多肽的膜穿透能力，从而促进细胞内基因传递。苄基（Bn）和萘基（Naph）修饰的多肽表现出最高的 DNA 摄取水平，比未修饰的多肽 P2 高约 4 倍。更重要的是，与 P2 相比，Bn 和 Naph 修饰的多肽允许更多的 DNA 货物通过非内吞途径内化，这显著绕过了内体捕获，从而将转染效率提高了 42 倍，比商用试剂 PEI 25k 高 3-4 个数量级。芳族修饰还提高了多肽的 siRNA 凝聚能力，实现了对肿瘤坏死因子-α的显著增强的基因沉默效率，从而治疗急性肝炎症。此外，我们揭示了芳香性增强的膜活性伴随着可比甚至显著降低的“孔形成”能力，从而在高浓度下降低了细胞毒性。因此，这项研究提供了一种有前途的方法来操纵聚阳离子的膜活性和穿透机制，克服了阻止有效和安全基因传递的多个关键障碍。
