title: Nuclear targeted delivery of macromolecules to retina and cornea.
abstract: BACKGROUND
  Cell-penetrating peptides (CPPs) can deliver molecules into cells by binding and penetrating the plasma membrane. However, the majority of CPPs get trapped in endosomes, resulting in degradation of the cargo molecule and inefficient delivery to the nucleus. The present study investigates the potential use of a nucleolin binding peptide (NBP) for the delivery of macromolecules including fluorophores, recombinant protein and DNA to the nuclei of ocular tissues in vivo.
  
  
  METHODS
  Fluorescent dyes covalently linked to NBP or NBP-green fluorescent protein fusion protein were injected intravitreally or subretinally or topically applied to the cornea. Frozen sections were prepared for quantification of transduction. Delivery of plasmid DNA was studied using luciferase and LacZ DNA compacted with pegylated NBP. Levels of luciferase were quantified, and LacZ expression was localized in ocular tissues.
  
  
  RESULTS
  We found that NBP-directed fluorophores exhibited retinal and corneal transduction. Subretinal injection transduced cell types throughout the retina, including photoreceptors, retinal pigment epithelium and neuronal cells. Intravitreal injection transduced neuronal cells in the retina, as well as cells in the cornea. Topically applied NBP lead to transduction of the superficial epithelial layer of the cornea. NBP localized to the nucleus upon exogenous application in vivo. Pegylated NBP nanoparticles significantly improved delivery and expression of transgenes over DNA alone without any measureable toxicity.
  
  
  CONCLUSIONS
  The results obtained in the present study demonstrate that NBP can deliver small and large molecules into retinal and corneal cells and plasmid DNA into retinal cells and hence may be useful for the delivery of therapeutics to the eye.
authors.affiliation: Department of Ophthalmology, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Binder
authors.forename: Sarah Parker
authors.initials: SP
authors.lastname: Read
authors.forename: Siobhan M
authors.initials: SM
authors.lastname: Cashman
authors.forename: Rajendra
authors.initials: R
authors.lastname: Kumar-Singh
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000287:Administration, Topical; D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D057846:Cell-Penetrating Peptides / Q000008:administration & dosage / Q000378:metabolism; D003315:Cornea / Q000378:metabolism*; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D016503:Drug Delivery Systems / Q000379:methods*; D004596:Electroretinography; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000378:metabolism; D049452:Green Fluorescent Proteins; D024242:HMGN2 Protein / Q000008:administration & dosage* / Q000378:metabolism; D006801:Humans; D058449:Intravitreal Injections; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D053758:Nanoparticles; D019913:Nuclear Localization Signals / Q000378:metabolism; D010455:Peptides / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010957:Plasmids / Q000008:administration & dosage / Q000378:metabolism; D011092:Polyethylene Glycols / Q000008:administration & dosage / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics; D000097796:Nucleolin
chemical_list: D057846:Cell-Penetrating Peptides; D004247:DNA; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D024242:HMGN2 Protein; D008156:Luciferases; D019913:Nuclear Localization Signals; D010455:Peptides; D010750:Phosphoproteins; D011092:Polyethylene Glycols; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D000097796:Nucleolin; C560441:nucleolin binding peptide