Cell binding, uptake and cytosolic partition of HIV anti-gag phosphodiester oligonucleotides 3 '-linked to cholesterol derivatives in macrophages

The purpose of this study is to evaluate the cell interactions of a new cla ss of compounds composed of phosphodiester oligonucleotides linked to the c holesterol group at position 3, 7, or 22 of the steroid structure, The resu lting conjugates were assessed for their capacity to bind, penetrate and pa rtition in the cytoplasmic compartment of murine macrophages. The results s howed that lipophilic conjugates bind to cells much faster (t(1/2) less tha n or equal to 10 min) than do underivatized oligomers. Oligomers tethered t o the cholesterol at positions 3 and 7 (PO-GEM-3-Chol and PO-GEM-7-Chol) in teracted more efficiently with cell membranes and were better internalized than oligomers attached to the cholesterol moiety at position 22 (PO-GEM-22 -Chol). The cytosolic fraction of internalized oligomers was studied by a d igitonin-based membrane permeabilization method. The recovered fraction of oligomers that can freely diffuse from the cytosol was comparable for GEM-9 1(TM), a phosphorothioate congener, and for PO-GEM-7-Chol (50-60% of the in ternalized oligomers), while that of PO-GEM-3-Chol was less (30% of the int ernalized oligomers) indicating a higher membrane affinity of the latter de rivative as compared to the other investigated compounds. Membrane binding and cell internalization correlated well with the hydrophobicity of the con jugates as characterized by their partition coefficients in a water-octanol system, Due to their capacity of rapid binding and cytosolic partition in cells, cholesterol-derivatized oligonucleotides at position 3 or 7 of the s teroid molecule appeared as good candidates for systemic delivery of anti-H IV antisense compounds. (C) 1999 Elsevier Science Ltd. All rights reserved.