Lin Ran, Zhang Pengcheng, Cheetham Andrew G, Walston Jeremy, Abadir Peter, Cui Honggang
Department of Chemical and Biomolecular Engineering, ‡Institute for NanoBioTechnology, §Division of Geriatrics Medicine and Gerontology, and ⊥Department of Oncology and Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University , Baltimore, Maryland 21218, United States.
Bioconjug Chem. 2015 Jan 21;26(1):71-7. doi: 10.1021/bc500408p. Epub 2014 Dec 30.
Mitochondria are critical regulators of cellular function and survival. Delivery of therapeutic and diagnostic agents into mitochondria is a challenging task in modern pharmacology because the molecule to be delivered needs to first overcome the cell membrane barrier and then be able to actively target the intracellular organelle. Current strategy of conjugating either a cell penetrating peptide (CPP) or a subcellular targeting sequence to the molecule of interest only has limited success. We report here a dual peptide conjugation strategy to achieve effective delivery of a non-membrane-penetrating dye 5-carboxyfluorescein (5-FAM) into mitochondria through the incorporation of both a mitochondrial targeting sequence (MTS) and a CPP into one conjugated molecule. Notably, circular dichroism studies reveal that the combined use of α-helix and PPII-like secondary structures has an unexpected, synergistic contribution to the internalization of the conjugate. Our results suggest that although the use of positively charged MTS peptide allows for improved targeting of mitochondria, with MTS alone it showed poor cellular uptake. With further covalent linkage of the MTS-5-FAM conjugate to a CPP sequence (R8), the dually conjugated molecule was found to show both improved cellular uptake and effective mitochondria targeting. We believe these results offer important insight into the rational design of peptide conjugates for intracellular delivery.
线粒体是细胞功能和存活的关键调节因子。将治疗和诊断药物递送至线粒体是现代药理学中的一项具有挑战性的任务，因为待递送的分子首先需要克服细胞膜屏障，然后能够主动靶向细胞内细胞器。目前将细胞穿透肽（CPP）或亚细胞靶向序列与感兴趣的分子偶联的策略仅取得了有限的成功。我们在此报告一种双肽偶联策略，通过将线粒体靶向序列（MTS）和CPP都整合到一个偶联分子中，实现将非膜穿透性染料5-羧基荧光素（5-FAM）有效递送至线粒体。值得注意的是，圆二色性研究表明，α-螺旋和类PPII二级结构的联合使用对偶联物的内化具有意想不到的协同作用。我们的结果表明，虽然使用带正电荷的MTS肽可改善线粒体靶向性，但单独使用MTS时细胞摄取较差。通过将MTS-5-FAM偶联物与CPP序列（R8）进一步共价连接，发现双偶联分子显示出改善的细胞摄取和有效的线粒体靶向性。我们相信这些结果为合理设计用于细胞内递送的肽偶联物提供了重要的见解。
