Antisense Effects of Cholesterol-Oligodeoxynucleotide Conjugates Associated with Poly(alkylcyanoacrylate) Nanoparticles

Abstract

Oligonucleotides covalently attached to a cholesteryl moiety are more stable in biological media and better taken up by eukaryotic cells. However, their anchoring in hydrophobic cellular membranes and endosomes after endocytosis restricts their access to cellular nucleic acids. New methods of cellular delivery and the biological activity of the conjugates were studied. The cholesteryl residue was conjugated via disulfide bond to the 5' or 3' terminal phosphate group of two oligodeoxyribonucleotide dodecamers complementary to the mutated region of Ha-ras oncogene mRNA. The conjugates were able to form complementary duplexes with the mutated 27-b target fragment of mRNA but not with the wild-type sequence. Efficient sequence-specific RNase H cleavage of complementary mRNA was induced with low (< or = 500 nM) concentrations of the conjugates. At higher concentrations, this cleavage was progressively inhibited, probably due to an interaction between RNase H and the cholesterol residue. The hydrophobic conjugates could be adsorbed onto poly(isohexylcyanoacrylate) nanoparticles via their cholesteryl moieties and delivered to eukaryotic cells. Cholesterol-conjugated oligonucleotides were able to sequence-specifically inhibit the proliferation of T24 human bladder carcinoma cells in culture.

Enhanced Hepatic Uptake and Bioactivity of Type α1(I) Collagen Gene Promoter-Specific Triplex-Forming Oligonucleotides after Conjugation with Cholesterol

“…Hydrophobization of ODNs is known to facilitate their binding to cellular membranes, enhance their cellular uptake by endocytosis, and improve their stability (Godard et al, 1995). In addition, conjugation with cholesterol has been reported to enhance accumulation of ODNs and siRNA in the liver, resulting in their improved biological activity (Godard et al, 1995;Bijsterbosch et al, 2000;Manoharan, 2004).…”

“…In addition, conjugation with cholesterol has been reported to enhance accumulation of ODNs and siRNA in the liver, resulting in their improved biological activity (Godard et al, 1995;Bijsterbosch et al, 2000;Manoharan, 2004). In this study, we conjugated cholesterol to type ␣1(I) collagen gene promoter-specific TFO at 3Ј end and characterized TFOChol in terms of triplex formation efficiency, cellular and subcellular uptake, bioactivity, tissue distribution, and hepatic cellular localization after systemic administration of 33 P-TFO-Chol in normal and fibrotic rats.…”

The use of synthetic polymers for delivery of therapeutic antisense oligodeoxynucleotides

“…Unlike the cationic polystyrene described above, the charges on the surface of PCAs are not part of the chemical structure of the polymer, as they are introduced in the system at the time of complexation with ODNs. These investigations were commenced by a group at the Centre National de la Recherche Scientifique in Paris, which provided most of the results currently available [151][152][153][154][155][156]. As mediators for ion-pair formation, hydrophobic cations such as quaternary ammonium salts or tetraphenylphosphomium chloride were proposed; in the majority of experiments, cetyltrimethylammonium bromide was added to ODN prior to complexation to the polymer nanoparticles.…”

New Technologies for Drug Delivery Across the Blood Brain Barrier

“…The use of nanoparticles as vehicles for drug and gene delivery has been an area of intensive research and development for over a decade (43)(44)(45)(46). Some examples, include solid nanoparticles (44,47,48), liposomes (49-51) and polymer micelles (52)(53)(54)(55)(56). The surface of such carriers is often modified by poly(ethylene glycol) (PEG) 1 brush ("PEGylation") to increase the stability of nanoparticles in dispersion and extend circulation time of nanoparticles in the body (46,(57)(58)(59).…”