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 αvβ3 integrin. We used αvβ3-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 αvβ3 integrin.

Dual-ligand modification of PEGylated liposomes shows better cell selectivity and efficient gene delivery

“…whether it is on the surface of the lipid membrane of LP or on the top of the PEG [43]. The RGD modified PEG-LP (RGD-PEG-LP) is taken up mainly by caveolae-mediated endocytosis, which is consistent with findings reported by other research groups [36,44]. Interestingly, it was observed that, after the incorporation of R8 and RGD into the PEG-LP, the internalization of R8/RGD-PEG-LP is predominantly governed by the clathrin-mediated endocytosis pathway.…”

The Biological Effect of an Antisense Oligonucleotide Depends on Its Route of Endocytosis and Trafficking

“…In both cases the fl uorescent oligonucleotide was primarily found in intracellular vesicles, similar to previous descriptions of the subcellular distribution of oligonucleotides and peptide-oligonucleotide conjugates observed by us and by others (Turner et al, 2005;Alam et al, 2008). Thus, we sought to probe possible differences in uptake and traffi cking pathways by use of several putatively selective inhibitors of various endocytotic pathways (Parpal et al, 2001;Khalil et al, 2006).…”

Reductive nanocomplex encapsulation of cRGD-siRNA conjugates for enhanced targeting to cancer cells

“…26 Conjugation of cyclic RGD with Sulfo-AC 5 -SPDP was performed as follows: RGD peptide (386 μM) was mixed with Sulfo-AC 5 -SPDP in PBS and incubated for 3 h at room temperature. The resulting product, cRGD-PDP, was analyzed and purified by using a HPLC with a C18 column (Venusil XBP C18 (2), 4.6×250 mm, 150 Å, 5 μm) equipped with a UV detector at 220 nm.…”

“…It has been previously demonstrated that A375 cell lines express integrin α v β 3 on their cell surface. 26,28 Therefore, A375 cell line was selected to explore the gene-silencing ability of CLD/cRGD-siRNA nanocomplexes in the following in vitro studies. The cells were cultured in DMEM in a humidified incubator supplied with 5% CO 2 and maintained at 37°C.…”