Multivalent Cyclic RGD Conjugates for Targeted Delivery of Small Interfering RNA

Abstract

We have designed, synthesized and tested conjugates of chemically modified luciferase siRNA (Luc-siRNA) with bi-, tri- and tetravalent cyclic(arginine-glycine-aspartic) peptides (cRGD) that selectively bind to the αvβ3 integrin. The cellular uptake, subcellular distribution and pharmacological effects of the cRGD conjugated Luc-siRNAs as compared to un-conjugated controls were examined using a luciferase reporter cassette stably transfected into αvβ3 positive M21+ human melanoma cells. The M21+ cells exhibited receptor-mediated uptake of cRGD-siRNA conjugates but not of unconjugated control siRNA. The fluorophore-tagged cRGD-siRNA conjugates were taken up by a caveolar endocytotic route and primarily accumulated in cytosolic vesicles. The bi-, tri- and tetravalent cRGD conjugates were taken up by M21+ cells to approximately the same degree. However, there were notable differences in their pharmacological effectiveness. The tri- and tetravalent versions produced progressive, dose-dependent reductions in luciferase expression, while the bivalent version had little effect. The basis for this divergence of uptake and effect is currently unclear. Nonetheless the high selectivity and substantial ‘knock down’ effects of the multivalent cRGD-siRNA conjugates suggest that this targeting and delivery strategy deserves further exploration.

A tumor-targeting cRGD-EGFR siRNA conjugate and its anti-tumor effect on glioblastoma in vitro and in vivo

“…To demonstrate the effect of cRGD binding to the avb3 receptor, the U87MG cells were also pre-treated with 1 mM of un-conjugated cRGD peptide (cRGD blocked) for 30 min at 37 C prior to transfection with cRGD-siEGFR-Cy5, as shown in a previous report (Alam et al, 2011). The final concentration of the compound was 100 nM.…”

Comparative Characterization of Hepatic Distribution and mRNA Reduction of Antisense Oligonucleotides Conjugated with Triantennary N-Acetyl Galactosamine and Lipophilic Ligands Targeting Apolipoprotein B

“…ASOs (Fig. 1) were synthesized using standard phosphoramidite chemistry with commercially available phosphoramidite monomers and postsynthetic modification (Alam et al, 2011). For oligo synthesis, cholesteryl-TEG phosphoramidite (Glen Research, Sterling, VA), Τoc-TEG phosphoramidite (Glen Research), and N-MMTraminohexyl linker phosphoramidite (Sigma-Aldrich) were used.…”

Orthogonal Protection of Peptides and Peptoids for Cyclization by the Thiol–Ene Reaction and Conjugation

“…Here the functional group for conjugation appended from a residue placed within the cycle. In one case the polyamide was a peptide and the functional group for conjugation a thiol (20), while in the other it was a peptoid derivatized with an alkyne (24).…”

“…19 Cyclic peptides have been covalently linked to the sense strand of a siRNA duplex, which facilitated transport of the oligonucleotide through the cell membrane and inhibited gene expression. 20 More recently, disulfide-containing citrullinated peptides have been linked to various oligonucleotides and immobilized on plates for diagnostic ELISA assays. 21 In this manuscript we wish to describe methodology allowing for the straightforward synthesis of [cyclic peptide]-oligonucleotide conjugates.…”