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Preparation of conjugates of oligodeoxynucleotides and lipid structures and their interaction with low-density
E T Rump1, R L de Vrueh, L A Sliedregt
1Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, The Netherlands.
Abstract:
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.
Insights
Researchers developed lipid-conjugated antisense oligodeoxynucleotides (ODNs) for targeted cancer therapy. These modified ODNs effectively associate with low-density lipoprotein (LDL) for delivery via the LDL receptor pathway.
Area of Science:
- Biochemistry
- Molecular Biology
- Nanotechnology
Background:
- Tumor cells overexpress low-density lipoprotein (LDL) receptors, presenting an opportunity for targeted drug delivery.
- Antisense oligodeoxynucleotides (ODNs) offer potential for oncogene-directed cancer therapy.
- Encapsulating ODNs within LDL could enhance their selective delivery to cancer cells.
Purpose of the Study:
- To conjugate oncogene-directed antisense ODNs with various lipids to facilitate their incorporation into LDL.
- To evaluate the association efficiency of lipid-conjugated ODNs with LDL for targeted delivery.
- To establish effective methods for purifying lipid-conjugated ODNs.
Main Methods:
- Synthesis of steroid lipids from bile acids with varying lipophilicity using oleic acid ester structures.
- Conjugation of lipid structures (activated as pentafluorophenyl esters) to 3'-amino-tailed ODNs.
- Purification of lipid-ODNs using reversed-phase HPLC and agarose gel electrophoresis with Tween 20.
- Assessment of ODN-LDL association using various lipid conjugation strategies.
Main Results:
- ODNs conjugated with lithocholic acid, oleic acid, and cholesterol were purified by RP-HPLC.
- Highly lipophilic ODNs conjugated with oleoyl steroid esters required purification by agarose gel electrophoresis.
- ODNs conjugated with cholesterol and specific oleoyl esters showed efficient association with LDL, while others showed incomplete or no association.
Conclusions:
- Several lipid-ODN conjugates demonstrate efficient association with LDL, enabling targeted delivery.
- Lipid conjugation strategies are crucial for successful incorporation into LDL for therapeutic applications.
- This approach facilitates the delivery of oncogene-directed antisense ODNs via the LDL receptor pathway for cancer treatment.