title: Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis.
abstract: We describe the synthesis and characterization of a 5' conjugate between a 2'-O-Me phosphorothioate antisense oligonucleotide and a bivalent RGD (arginine-glycine-aspartic acid) peptide that is a high-affinity ligand for the alphavbeta3 integrin. We used alphavbeta3-positive melanoma cells transfected with a reporter comprised of the firefly luciferase gene interrupted by an abnormally spliced intron. Intranuclear delivery of a specific antisense oligonucleotide (termed 623) corrects splicing and allows luciferase expression in these cells. The RGD-623 conjugate or a cationic lipid-623 complex produced significant increases in luciferase expression, while 'free' 623 did not. However, the kinetics of luciferase expression was distinct; the RGD-623 conjugate produced a gradual increase followed by a gradual decline, while the cationic lipid-623 complex caused a rapid increase followed by a monotonic decline. The subcellular distribution of the oligonucleotide delivered using cationic lipids included both cytoplasmic vesicles and the nucleus, while the RGD-623 conjugate was primarily found in cytoplasmic vesicles that partially co-localized with a marker for caveolae. Both the cellular uptake and the biological effect of the RGD-623 conjugate were blocked by excess RGD peptide. These observations suggest that the bivalent RGD peptide-oligonucleotide conjugate enters cells via a process of receptor-mediated endocytosis mediated by the alphavbeta3 integrin.
authors: Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill NC 27599, USA.
authors: Md Rowshon
authors: MR
authors: Alam
authors: Vidula
authors: V
authors: Dixit
authors: Hyunmin
authors: H
authors: Kang
authors: Zi-Bo
authors: ZB
authors: Li
authors: Xiaoyuan
authors: X
authors: Chen
authors: Joann
authors: J
authors: Trejo
authors: Michael
authors: M
authors: Fisher
authors: Rudy L
authors: RL
authors: Juliano
mesh_terms: D045744:Cell Line, Tumor; D004705:Endocytosis; D017930:Genes, Reporter; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D007700:Kinetics; D049409:Luciferases, Firefly / Q000032:analysis / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000032:analysis / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry*
chemical_list: D039302:Integrin alphaVbeta3; D016376:Oligonucleotides, Antisense; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid; D049409:Luciferases, Firefly