Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong , 21 Sassoon Road, Pokfulam, Hong Kong.
Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong , 21 Sassoon Road, Pokfulam, Hong Kong.
Mol Pharm. 2017 Dec 4;14(12):4606-4617. doi: 10.1021/acs.molpharmaceut.7b00725. Epub 2017 Nov 17.
Pulmonary delivery of small interfering RNA (siRNA) has huge potential for the treatment of a wide range of respiratory diseases. The ability of naked siRNA to transfect cells in the lungs without a delivery vector has prompted the investigation of whether an endogenous component is at least partially responsible for the cellular uptake of siRNA, and whether a safe and efficient delivery system could be developed from this component to further improve the transfection efficiency. Surfactant protein B (SP-B), a positively charged protein molecule found in lung surfactant, is one of the possible candidates. While the role of SP-B in siRNA transfection remains to be determined, the SP-B mimic, synthetic KL4 peptide, was investigated in this study as a potential siRNA carrier. KL4 is a 21-residue cationic peptide that was able to bind to siRNA to form nanosized complexes. It mediated siRNA transfection effectively in vitro on human lung epithelial cells, A549 cells, and BEAS-2B cells, which was comparable to Lipofectamine 2000. When commercial pulmonary surfactant (Infasurf) was added in the transfection medium, the gene silencing effect of siRNA in cells transfected with Lipofectamine 2000 was completely abolished, whereas those transfected with KL4 remained unaffected. At 4 °C, KL4 failed to deliver siRNA into the cells, indicating that an energy-dependent process was involved in the uptake of the complexes. Chlorpromazine (inhibitor of chathrin-mediated endocytosis), but not nystatin (inhibitor of caveolae-mediated endocytosis), inhibited the uptake of KL4/siRNA complexes, suggesting that they entered cells through clathrin-mediated endocytosis. There was no sign of cytotoxicity or immune response caused by KL4 and KL4/siRNA complexes. Overall, this study demonstrated that synthetic KL4 peptide is a promising candidate for siRNA carrier for pulmonary delivery and could be a potential platform for delivering other types of nucleic acid therapeutics.
肺部递送小干扰 RNA（siRNA）在治疗广泛的呼吸道疾病方面具有巨大潜力。由于裸露的 siRNA 能够在没有递送载体的情况下转染肺部细胞，因此促使人们研究内源性成分是否至少部分负责 siRNA 的细胞摄取，以及是否可以从该成分开发出安全有效的递送系统，以进一步提高转染效率。表面活性蛋白 B（SP-B）是肺表面活性物质中带正电荷的蛋白质分子之一，是可能的候选物之一。尽管 SP-B 在 siRNA 转染中的作用仍有待确定，但本研究中研究了 SP-B 类似物，即合成 KL4 肽，作为潜在的 siRNA 载体。KL4 是一种 21 个残基的阳离子肽，能够与 siRNA 结合形成纳米大小的复合物。它在体外有效地介导了人肺上皮细胞、A549 细胞和 BEAS-2B 细胞中的 siRNA 转染，与 Lipofectamine 2000 相当。当在转染培养基中添加商业肺表面活性剂（Infasurf）时，用 Lipofectamine 2000 转染的细胞中的 siRNA 的基因沉默效果完全被废除，而用 KL4 转染的细胞则不受影响。在 4°C 下，KL4 未能将 siRNA 递送至细胞内，表明摄取复合物涉及能量依赖性过程。氯丙嗪（网格蛋白介导的内吞作用抑制剂），而不是制霉菌素（小窝蛋白介导的内吞作用抑制剂），抑制 KL4/siRNA 复合物的摄取，表明它们通过网格蛋白介导的内吞作用进入细胞。KL4 和 KL4/siRNA 复合物没有引起细胞毒性或免疫反应的迹象。总的来说，这项研究表明，合成 KL4 肽是肺部递送 siRNA 的有前途的载体候选物，并且可能是递送其他类型核酸治疗药物的潜在平台。
