Delivery of cholesteryl-conjugated phosphorothioate oligodeoxynucleotides to Kupffer cells by lactosylated low-density lipoprotein

The efficacy of antisense oligonucleotides depends on the ability to reach in vivo their target cells. We aim to develop strategies to enhance uptake of phosphorothioate oligodeoxynucleotides by Kupffer cells. To this end, we conjugated cholesterol to ISIS-3082, a phosphorothioate oligodeoxynucleoti de specific for intercellular adhesion molecule-1. The cholesterol-conjugat ed oligonucleotide, denoted ISIS-9388, associated readily with lactosylated low-density lipoprotein (LacLDL), a lipidic carrier that is taken up by ga lactose receptors on Kupffer cells. Association of up to 10 molecules of IS IS-9388 per LacLDL particle did not induce aggregation. LacLDL-associated [ H-3]ISIS-9388 was rapidly taken up by the liver after injection into rats ( 52.9 +/- 1.8% of the dose within 2 min versus 18.6 +/- 2.8% for ISIS-3082). N-acetylgalactosamine inhibited hepatic uptake, indicating involvement of galactose-specific receptors. Liver cells were isolated at 60 min after inj ection of LacLDL-associated [H-3]ISIS-9388. Kupffer cells displayed the hig hest uptake: 88.1 +/- 24.7 ng of oligonucleotide/mg of cell protein, which is 6-14 times higher than after injection of free ISIS-9388 or ISIS-3082 (1 5.0 +/- 3.8 ng and 6.3 +/- 1.4 ng, respectively). It can be calculated that Kupffer cells contribute 43.9 +/- 5.4% to the Ever uptake (free ISIS-9388 and ISIS-3083 14.5 +/- 3.1% and 8.3 +/- 3.2%, respectively). In conclusion, conjugation of a phosphorothioate oligodeoxynucleotide with cholesterol an d its subsequent association with LacLDL results in a substantially increas ed Kupffer cell uptake of the oligonucleotide. As Kupffer cells play a key role in inflammation, our approach may be utilized to improve antisense-bas ed therapeutic intervention during inflammation. (C) 2001 Elsevier Science Inc. All rights reserved.