title: A Redox-Activatable Fluorescent Sensor for the High-Throughput Quantification of Cytosolic Delivery of Macromolecules.
abstract: Efficient delivery of biomacromolecules (e.g., proteins, nucleic acids) into cell cytosol remains a critical challenge for the development of macromolecular therapeutics or diagnostics. To date, most common approaches to assess cytosolic delivery rely on fluorescent labeling of macromolecules with an "always on" reporter and subcellular imaging of endolysosomal escape by confocal microscopy. This strategy is limited by poor signal-to-noise ratio and only offers low throughput, qualitative information. Herein we describe a quantitative redox-activatable sensor (qRAS) for the real-time monitoring of cytosolic delivery of macromolecules. qRAS-labeled macromolecules are silent (off) inside the intact endocytic organelles, but can be turned on by redox activation after endolysosomal disruption and delivery into the cytosol, thereby greatly improving the detection accuracy. In addition to confocal microscopy, this quantitative sensing technology allowed for a high-throughput screening of a panel of polymer carriers toward efficient cytosolic delivery of model proteins on a plate reader. The simple and versatile qRAS design offers a useful tool for the investigation of new strategies for endolysosomal escape of biomacromolecules to facilitate the development of macromolecular therapeutics for a variety of disease indications.
authors.affiliation: Department of Pharmacology, Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA.
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Pharmacology, Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Luo
authors.affiliation: Department of Pharmacology, Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA.
authors.forename: Chengqiong
authors.initials: C
authors.lastname: Mao
authors.affiliation: Department of Pharmacology, Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wei
authors.affiliation: Department of Pharmacology, Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA.
authors.forename: Tian
authors.initials: T
authors.lastname: Zhao
authors.affiliation: Department of Pharmacology, Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Pharmacology, Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Huang
authors.affiliation: Department of Pharmacology, Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA.
authors.forename: Jinming
authors.initials: J
authors.lastname: Gao
mesh_terms: D000072283:A549 Cells; D003600:Cytosol / Q000378:metabolism*; D016503:Drug Delivery Systems / Q000379:methods*; D011992:Endosomes / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D005978:Glutathione; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D010720:Phosphines; C080938:tris(2-carboxyethyl)phosphine
keywords: FRET; activatable sensors; drug delivery; endolysosomal disruption; high-throughput screening