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Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis.

Alam MR, Dixit V, Kang H, Li ZB, Chen X, Trejo J, Fisher M, Juliano RL - Nucleic Acids Res. (2008)

Bottom Line: The RGD-623 conjugate or a cationic lipid-623 complex produced significant increases in luciferase expression, while 'free' 623 did not.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill NC 27599, USA.

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.

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Time–response studies. Cells were treated with either 623-Tamra, RGD–623-Tamra conjugate or 623-Tamra complexed with Lipofectamine 2000, as described in Materials and methods section, and luciferase activity was determined at the times indicated. Black bars represent luciferase activity of 200 nM 623-Tamra, patterned/striped bars represent 200 nM RGD–623-Tamra conjugate and gray bars represent 100 nM 623-Tamra transfected using Lipofectamine 2000, all expressed as RLUs per 105 cells. Results are the means and standard errors of triplicate determinations.
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Figure 3: Time–response studies. Cells were treated with either 623-Tamra, RGD–623-Tamra conjugate or 623-Tamra complexed with Lipofectamine 2000, as described in Materials and methods section, and luciferase activity was determined at the times indicated. Black bars represent luciferase activity of 200 nM 623-Tamra, patterned/striped bars represent 200 nM RGD–623-Tamra conjugate and gray bars represent 100 nM 623-Tamra transfected using Lipofectamine 2000, all expressed as RLUs per 105 cells. Results are the means and standard errors of triplicate determinations.

Mentions: We examined the kinetics and duration of action of the RGD–623 conjugate by harvesting the cells at various times after the period of exposure to the oligonucleotide. As seen in Figure 3, there was a striking difference between the kinetics of the RGD–623 conjugate and the cationic lipid/623 complex. Thus, the effect of the RGD–623 conjugate on luciferase expression rose gradually with time and reached a maximum at 72 h (48 h after removal of the oligonucleotide). In contrast, the effect of the cationic lipid/623 complex was greatest at very early time points after exposure to the oligonucleotide and declined steadily thereafter. This indicates that the two modes of delivery operate by very different mechanisms. The oligonucleotide delivered via cationic lipids seems to rapidly go to the nucleus, while that delivered via the peptide-conjugate seems to traffic through other intracellular compartments and only gradually reach the nucleus where the effect on splicing takes place.Figure 3.


Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis.

Alam MR, Dixit V, Kang H, Li ZB, Chen X, Trejo J, Fisher M, Juliano RL - Nucleic Acids Res. (2008)

Time–response studies. Cells were treated with either 623-Tamra, RGD–623-Tamra conjugate or 623-Tamra complexed with Lipofectamine 2000, as described in Materials and methods section, and luciferase activity was determined at the times indicated. Black bars represent luciferase activity of 200 nM 623-Tamra, patterned/striped bars represent 200 nM RGD–623-Tamra conjugate and gray bars represent 100 nM 623-Tamra transfected using Lipofectamine 2000, all expressed as RLUs per 105 cells. Results are the means and standard errors of triplicate determinations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2377441&req=5

Figure 3: Time–response studies. Cells were treated with either 623-Tamra, RGD–623-Tamra conjugate or 623-Tamra complexed with Lipofectamine 2000, as described in Materials and methods section, and luciferase activity was determined at the times indicated. Black bars represent luciferase activity of 200 nM 623-Tamra, patterned/striped bars represent 200 nM RGD–623-Tamra conjugate and gray bars represent 100 nM 623-Tamra transfected using Lipofectamine 2000, all expressed as RLUs per 105 cells. Results are the means and standard errors of triplicate determinations.
Mentions: We examined the kinetics and duration of action of the RGD–623 conjugate by harvesting the cells at various times after the period of exposure to the oligonucleotide. As seen in Figure 3, there was a striking difference between the kinetics of the RGD–623 conjugate and the cationic lipid/623 complex. Thus, the effect of the RGD–623 conjugate on luciferase expression rose gradually with time and reached a maximum at 72 h (48 h after removal of the oligonucleotide). In contrast, the effect of the cationic lipid/623 complex was greatest at very early time points after exposure to the oligonucleotide and declined steadily thereafter. This indicates that the two modes of delivery operate by very different mechanisms. The oligonucleotide delivered via cationic lipids seems to rapidly go to the nucleus, while that delivered via the peptide-conjugate seems to traffic through other intracellular compartments and only gradually reach the nucleus where the effect on splicing takes place.Figure 3.

Bottom Line: The RGD-623 conjugate or a cationic lipid-623 complex produced significant increases in luciferase expression, while 'free' 623 did not.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill NC 27599, USA.

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.

Show MeSH
Related in: MedlinePlus