title: Internalization of novel non-viral vector TAT-streptavidin into human cells.
abstract: BACKGROUND
  The cell-penetrating peptide derived from the Human immunodeficiency virus-1 transactivator protein Tat possesses the capacity to promote the effective uptake of various cargo molecules across the plasma membrane in vitro and in vivo. The objective of this study was to characterize the uptake and delivery mechanisms of a novel streptavidin fusion construct, TAT47-57-streptavidin (TAT-SA, 60 kD). SA represents a potentially useful TAT-fusion partner due to its ability to perform as a versatile intracellular delivery vector for a wide array of biotinylated molecules or cargoes.
  
  
  RESULTS
  By confocal and immunoelectron microscopy the majority of internalized TAT-SA was shown to accumulate in perinuclear vesicles in both cancer and non-cancer cell lines. The uptake studies in living cells with various fluorescent endocytic markers and inhibiting agents suggested that TAT-SA is internalized into cells efficiently, using both clathrin-mediated endocytosis and lipid-raft-mediated macropinocytosis. When endosomal release of TAT-SA was enhanced through the incorporation of a biotinylated, pH-responsive polymer poly(propylacrylic acid) (PPAA), nuclear localization of TAT-SA and TAT-SA bound to biotin was markedly improved. Additionally, no significant cytotoxicity was detected in the TAT-SA constructs.
  
  
  CONCLUSIONS
  This study demonstrates that TAT-SA-PPAA is a potential non-viral vector to be utilized in protein therapeutics to deliver biotinylated molecules both into cytoplasm and nucleus of human cells.
authors.affiliation: NanoScience Center; Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylä, Finland. johrinn@cc.jyu.fi <johrinn@cc.jyu.fi>
authors.forename: Johanna
authors.initials: J
authors.lastname: Rinne
authors.forename: Brian
authors.initials: B
authors.lastname: Albarran
authors.forename: Juulia
authors.initials: J
authors.lastname: Jylhävä
authors.forename: Teemu O
authors.initials: TO
authors.lastname: Ihalainen
authors.forename: Pasi
authors.initials: P
authors.lastname: Kankaanpää
authors.forename: Vesa P
authors.initials: VP
authors.lastname: Hytönen
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Stayton
authors.forename: Markku S
authors.initials: MS
authors.lastname: Kulomaa
authors.forename: Maija
authors.initials: M
authors.lastname: Vihinen-Ranta
mesh_terms: D016503:Drug Delivery Systems / Q000379:methods*; D015696:Gene Products, tat / Q000235:genetics / Q000493:pharmacokinetics*; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics; D019809:Streptavidin / Q000235:genetics / Q000493:pharmacokinetics*; D014780:Viruses / Q000235:genetics
chemical_list: D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D019809:Streptavidin