title: Dual peptide conjugation strategy for improved cellular uptake and mitochondria targeting.
abstract: 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.
authors.affiliation: 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.
authors.forename: Ran
authors.initials: R
authors.lastname: Lin
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Zhang
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Cheetham
authors.forename: Jeremy
authors.initials: J
authors.lastname: Walston
authors.forename: Peter
authors.initials: P
authors.lastname: Abadir
authors.forename: Honggang
authors.initials: H
authors.lastname: Cui
mesh_terms: D000595:Amino Acid Sequence; D057846:Cell-Penetrating Peptides / Q000737:chemistry* / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D021381:Protein Transport
chemical_list: D057846:Cell-Penetrating Peptides; D005452:Fluoresceins; C045132:4-carboxyfluorescein