title: Precise quantification of cellular uptake of cell-penetrating peptides using fluorescence-activated cell sorting and fluorescence correlation spectroscopy.
abstract: Cell-penetrating peptides (CPPs) have emerged as a potentially powerful tool for drug delivery due to their ability to efficiently transport a whole host of biologically active cargoes into cells. Although concerted efforts have shed some light on the cellular internalization pathways of CPPs, quantification of CPP uptake has proved problematic. Here we describe an experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules. This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane.
authors.affiliation: Biology Program, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates.
authors.forename: Rachid
authors.initials: R
authors.lastname: Rezgui
authors.affiliation: Physics Program, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates.
authors.forename: Katy
authors.initials: K
authors.lastname: Blumer
authors.affiliation: Center for Genomics and Systems Biology (CGSB), New York University Abu Dhabi, Abu Dhabi, United Arab Emirates.
authors.forename: Gilbert
authors.initials: G
authors.lastname: Yeoh-Tan
authors.affiliation: Laboratory of Molecular Biophysics, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Trexler
authors.affiliation: Biology Program, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates. Electronic address: mazin.magzoub@nyu.edu.
authors.forename: Mazin
authors.initials: M
authors.lastname: Magzoub
mesh_terms: D000643:Ammonium Chloride / Q000494:pharmacology; D001710:Biotin / Q000737:chemistry; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000032:analysis* / Q000378:metabolism; D002746:Chlorpromazine / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D005372:Filipin / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D021381:Protein Transport / Q000187:drug effects; D013050:Spectrometry, Fluorescence / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: C492694:Alexa Fluor 488 carboxylic acid succinimidyl ester; D057846:Cell-Penetrating Peptides; D005456:Fluorescent Dyes; D013388:Succinimides; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D000643:Ammonium Chloride; D015638:Cytochalasin D; D001710:Biotin; D005372:Filipin; D019809:Streptavidin; D002746:Chlorpromazine
keywords: Cellular uptake; Drug delivery; Internalization pathways; Intracellular degradation; Intracellular distribution; Toxicity threshold