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 oligodeoxynucleotide specific for intercellular adhesion molecule-1. The cholesterol-conjugated oligonucleotide, denoted ISIS-9388, associated readily with lactosylated low-density lipoprotein (LacLDL), a lipidic carrier that is taken up by galactose receptors on Kupffer cells. Association of up to 10 molecules of ISIS-9388 per LacLDL particle did not induce aggregation. LacLDL-associated [3H]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 injection of LacLDL-associated [3H]ISIS-9388. Kupffer cells displayed the highest 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 (15.0+/-3.8 ng and 6.3+/-1.4 ng, respectively). It can be calculated that Kupffer cells contribute 43.9+/-5.4% to the liver 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 and its subsequent association with LacLDL results in a substantially increased Kupffer cell uptake of the oligonucleotide. As Kupffer cells play a key role in inflammation, our approach may be utilized to improve antisense-based therapeutic intervention during inflammation.

Modification of the plasma clearance and liver uptake of steroid ester-conjugated oligodeoxynucleotides by association with (lactosylated) low-density lipoprotein.

Low-density lipoprotein (LDL) has been proposed as carrier for the selective delivery of anticancer drugs to tumor cells. We reported earlier the association of several lipidic steroid-conjugated anticancer oligodeoxynucleotides (ODNs) with LDL. In the present study, we determined the stability of these complexes. When the complexes were incubated with a mixture of high-density lipoprotein and albumin, or with rat plasma, the oleoyl steroid-conjugated ODNs appeared to be more stably associated with LDL than the cholesteryl-conjugated ODN. Intravenously injected free lipid-ODNs were very rapidly cleared from the circulation of rats. The area under the curve (AUC) of the lipid-ODNs in plasma was <0.4 microg x min/mL. After complexation with LDL, plasma clearance of the lipid-ODNs was delayed. This was most evident for ODN-5, the ODN conjugated with the oleoyl ester of lithocholic acid (AUC = 6.82 +/- 1.34 microg x min/mL). The AUC of ODN-4, a cholesteryl-conjugated ODN, was 1.49 +/- 0.37 microg x min/mL. In addition, the liver uptake of the LDL-complexed lipid-ODNs was reduced. The lipid-ODNs were also administered as a complex with lactosylated LDL, a modified LDL particle that is selectively taken up by the liver. A high proportion of ODN-5 was transported to the liver along with lactosylated LDL (69.1 +/- 8.1% of the dose at 15 min after injection), whereas much less ODN-4 was transported (36.6 +/- 0.1% of the dose at 15 min after injection). We conclude that the oleoyl ester of lithocholic acid is a more potent lipid anchor than the other steroid lipid anchors. Because of the stable association, the oleoyl ester of lithocholic acid is an interesting candidate for tumor targeting of anticancer ODNs with lipoproteins.