title: Methods to follow intracellular trafficking of cell-penetrating peptides.
abstract: Cell-penetrating peptides (CPPs) are efficient vehicles to transport bioactive molecules into the cells. Despite numerous studies the exact mechanism by which CPPs facilitate delivery of cargo to its intracellular target is still debated. The current work presents methods that can be used for tracking CPP/pDNA complexes through endosomal transport and show the role of endosomal transport in the delivery of cargo. Separation of endosomal vesicles by differential centrifugation enables to pinpoint the localization of delivered cargo without labeling it and gives important quantitative information about pDNA trafficing in certain endosomal compartments. Single particle tracking (SPT) allows following individual CPP/cargo complex through endosomal path in live cells, using fluoresently labled cargo and green fluoresent protein expressing cells. These two different methods show similar results about tested NickFect/pDNA complexes intracellular trafficing. NF51 facilitates rapid internalization of complexes into the cells, prolongs their stay in early endosomes and promotes release to cytosol. NF1 is less capable to induce endosomal release and higher amount of complexes are routed to lysosomes for degradation. Our findings offer potential delivery vector for in vivo applications, NF51, where endosomal entrapment has been allayed. Furthermore, these methods are valuable tools to study other CPP-based delivery systems.
authors.affiliation: a Laboratory of Molecular Biotechnology , Institute of Technology, University of Tartu , Tartu , Estonia .
authors.forename: Ly
authors.initials: L
authors.lastname: Pärnaste
authors.affiliation: a Laboratory of Molecular Biotechnology , Institute of Technology, University of Tartu , Tartu , Estonia .
authors.forename: Piret
authors.initials: P
authors.lastname: Arukuusk
authors.affiliation: b Laboratory for General Biochemistry and Physical Pharmacy , Ghent, University , Gent , Belgium , and.
authors.forename: Elisa
authors.initials: E
authors.lastname: Zagato
authors.affiliation: b Laboratory for General Biochemistry and Physical Pharmacy , Ghent, University , Gent , Belgium , and.
authors.forename: Kevin
authors.initials: K
authors.lastname: Braeckmans
authors.affiliation: a Laboratory of Molecular Biotechnology , Institute of Technology, University of Tartu , Tartu , Estonia .|c Department of Neurochemistry , The Arrhenius Laboratories for Natural Sciences, Stockholm University , Stockholm , Sweden.
authors.forename: Ülo
authors.initials: Ü
authors.lastname: Langel
mesh_terms: D057846:Cell-Penetrating Peptides / Q000008:administration & dosage* / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000008:administration & dosage*; D011992:Endosomes / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000008:administration & dosage / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D010957:Plasmids / Q000008:administration & dosage; D021381:Protein Transport
chemical_list: D057846:Cell-Penetrating Peptides; D049452:Green Fluorescent Proteins; D004247:DNA
keywords: Endocytosis; endosomal release