Preparation of conjugates of oligodeoxynucleotides and lipid structures and their interaction with low-density lipoprotein.
The high expression level of receptors for low-density lipoprotein (LDL) on tumor cells makes LDL an attractive carrier for selective delivery of drugs to these cells. The aim of this study is to allow incorporation of oncogene-directed antisense oligodeoxynucleotides (ODNs) into the lipid moiety of LDL. Therefore, ODNs were conjugated with oleic acid, cholesterol, and several other steroid lipids. These latter steroid lipids were synthesized starting from bile acids and were varied in lipophilicity by attaching oleic acid ester structures. The lipid structures, activated as pentafluorophenyl esters, were conjugated in solution phase to 3'-amino-tailed ODNs. The ODNs conjugated with lithocholic acid, oleic acid, and cholesterol could easily be purified by reversed phase (RP)-HPLC. However, the ODNs conjugated with the oleoyl steroid ester structures irreversibly bound to the column material. These highly lipidic ODNs were separated from the nonconjugated ODN by electrophoresis in a 1% low-melting agarose gel containing 0.1% Tween 20. This method was found to be very effective in isolating the ODNs conjugated to the oleoyl steroid ester structures. The ODNs conjugated with cholesterol and the oleoyl esters of lithocholic and cholenic acid associated readily and nearly completely with LDL. However, the less lipidic ODNs and the ODN conjugated with the dioleoyl ester of chenodeoxycholic acid did not and did incompletely associate, respectively. Lithocholic acid and oleic acid are probably not sufficiently lipophilic to induce association with LDL, whereas the dioleoyl ester structure is probably too bulky and extended to allow partitioning into the lipid moiety of LDL. We conclude that several of the lipid-ODNs can associate readily with LDL, enabling delivery of oncogene-directed antisense ODNs via the LDL receptor pathway.
Rump
E T
ET
Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, The Netherlands.
de Vrueh
R L
RL
Sliedregt
L A
LA
Biessen
E A
EA
van Berkel
T J
TJ
Bijsterbosch
M K
MK
D001647:Bile Acids and Salts / Q000737:chemistry; D002784:Cholesterol / Q000031:analogs & derivatives; D004587:Electrophoresis, Agar Gel; D008055:Lipids / Q000737:chemistry*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D015394:Molecular Structure; D009829:Oleic Acids / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D016376:Oligonucleotides, Antisense / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000737:chemistry; D020598:Proto-Oncogene Proteins c-myb; D013256:Steroids / Q000737:chemistry; D015534:Trans-Activators / Q000737:chemistry
D001647:Bile Acids and Salts; D008055:Lipids; D008077:Lipoproteins, LDL; D009829:Oleic Acids; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D013256:Steroids; D015534:Trans-Activators; D002784:Cholesterol