Preparation of Conjugates of Oligodeoxynucleotides and Lipid

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Bioconjugate Chem. 1998, 9, 341−349

341

Preparation of Conjugates of Oligodeoxynucleotides and Lipid Structures and Their Interaction with Low-Density Lipoprotein Erik T. Rump, Remco L. A. de Vrueh, Leo A. J. M. Sliedregt, Erik A. L. Biessen, Theo J. C. van Berkel, and Martin K. Bijsterbosch* Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, P.O. Box 9503, 2300 RA Leiden, The Netherlands. Received October 3, 1997; Revised Manuscript Received January 16, 1998

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 oncogenedirected antisense ODNs via the LDL receptor pathway.

INTRODUCTION

The so-called first-generation antisense ODNs,1 mostly phosphorothiate ODNs, were primarily designed to stabilize the nuclease-sensitive phosphodiester backbone of unmodified ODNs. Numerous reports have shown their effectiveness both in vitro and in vivo (1, 2). The success in preclinical research has led to approval of several antisense ODNs for clinical trials (2). Although the phosphorothioate ODNs and other backbone-modified ODNs are very promising in antisense research, cellular uptake of these modified ODNs still remains an inefficient process. Lipophilic moieties, conjugated to ODNs at either the 3′- or the 5′-end, stabilize the ODN and significantly enhance their cellular uptake (3-6). This can subsequently lead to improved antisense inhibition, as has been shown for cholesteryl-derivatized ODNs (7-11). The increased antisense efficacy of these ODNs may be a direct result of the affinity of the lipophilic moiety for membrane structures leading to a more efficient uptake, probably via a process of adsorptive endocytosis (4, 8, 10). * Author to whom correspondence should be addressed. E-mail: [email protected]. Telephone: 31-715276038. Fax: 31-71-5276032. 1 Abbreviations: BAP, 4-bromoacetophenyl; CH CN, aceto3 nitrile; DCC, dicyclohexylcarbodiimide; DCE, 1,2-dichloroethane; DCM, dichloromethane; DiPEA, N,N-diisopropylethylamine; DMAP, (dimethylamino)pyridine; DMF, N,N-dimethylformamide; ODN, oligodeoxynucleotide; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; Tm, melting temperature.

Alternatively, binding to the low-density lipoprotein (LDL) may also play a role, as it has been demonstrated that upregulation of the LDL receptor (LDLr) on cells increases the cellular uptake and effectiveness of cholesteryl-derivatized antisense ODNs (10). LDL is the main cholesterol-transporting vehicle in the human circulatory system. It is a spherical particle with a mean diameter of 23 nm, and it consists of an apolar core of cholesteryl esters and triglycerides, which is surrounded by a shell of cholesterol and phospholipids (12). A large part of the surface is covered by apolipoprotein B100, which is responsible for recognition by the LDLr. Binding of LDL to its receptor is followed by internalization of the particle. There is evidence that many malignant cells, like leukemia cells, take up large amounts of LDL via the LDLr. The LDL particle may therefore constitute an attractive carrier for selective and highly efficient intracellular delivery of antineoplastic drugs to tumor cells. The LDLr-mediated therapeutic approach has already been explored for conventional lipophilic antitumor drugs (13-16) but may also be applied for lipophilic oncogene-directed antisense ODNs. Earlier studies established the potential of receptormediated endocytosis to mediate effective internalization of ODNs, thereby increasing the antisense efficacy (17, 18). We and others have shown before that attachment of a cholesteryl moiety to the 5′-end allows association of ODNs with LDL in vitro. Association presumably occurs by partitioning of the relatively rigid cholesteryl moiety into the lipoprotein’s lipid shell (10, 19). In analogy,

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342 Bioconjugate Chem., Vol. 9, No. 3, 1998

conjugation of an alkyl chain was also shown to induce association of ODNs to LDL (20). In this study, we examine the ability of several lipophilic moieties to induce spontaneous association of ODNs to LDL. c-myb antisense ODNs were conjugated in solution phase at the 3′-end with lipidic structures which are composed of oleic acid and steroids. The antisense ODNs used in this study are directed to the c-myb proto-oncogene which is abundantly expressed in leukemic cells (21, 22). Numerous reports already showed the therapeutic potential of these ODNs in leukemic malignancies (23-25). Association of the lipid-ODNs to LDL is anticipated to enhance the cellular uptake, leading to an improved efficacy of the antisense ODN. EXPERIMENTAL SECTION

Reagents. An 18-mer antisense ODN complementary to the c-myb proto-oncogene (5′-G*T*G* CCG GGG TCT TCG GGC-3′), provided with a seven-carbon 3′-amine linker and three phosphorothioate linkages (*) at the 5′end, and a corresponding 28-mer sense ODN (5′-CCA TGG CCC GAA GAC CCC GGC ACA GCA T-3′) were from Eurogentec (Seraing, Belgium). Low-melting multipurpose (LM-MP) agarose and Agarase from Pseudomonas atlantica were obtained from Boehringer (Mannheim, Germany). Tween 20 was from Merck (Darmstadt, Germany). Hionic Fluor and Emulsifier Safe scintillation cocktails and Soluene-350 were from Packard (Downers Grove, IL). Pyridine and DCE were dried by refluxing with CaH2 and were distilled. DMF was stirred with CaH2 prior to distillation under reduced pressure. These and other solvents (analytical grade) were stored over molecular sieves (4 Å). Oleoyl chloride was distilled under reduced pressure before use. Na125I (carrier-free) and 3H2O were purchased from Amersham (Buckinghamshire, U.K.). DiPEA (99.5%, redistilled) was purchased from Aldrich (Milwaukee, WI). All other chemicals were of analytical grade. General Procedures. ODN concentrations were determined by UV spectroscopy at 260 nm. All reactions were run at ambient temperature, unless otherwise stated. Reactions were monitored by TLC on silica gel 60 F254 aluminum sheets (Merck). Compounds were visualized under UV (254 nm) or by spraying with MnCl2 for detection of steroids (26) or cresol green for carboxylic acids. Short column chromatography was performed on silica gel 60 (Merck, 230-400 mesh). 1H NMR spectra were measured at 200 MHz using a JEOL JNM-FX 200 spectrometer with tetramethylsilane as an internal standard. 13C NMR spectra were measured at 50.3 MHz on the same apparatus, with CDCl3 as an internal standard. 13 C NMR spectra were monitored using the attached proton test technique. 19F NMR spectra were measured at 282 MHz with CFCl3 as an internal standard on a Bruker WM-300 spectrometer. FAB mass spectrometry was carried out using a JEOL JMS SX/SX-102A foursector mass spectrometer. 5-Cholenic Acid 3β-Ol (4-Bromobenzoyl)methyl Ester 1b. To a solution of 250 mg (0.67 mmol) of cholenic acid 1a in 10 mL of DMF were added 205 mg (0.74 mmol) of dibromoacetophenone and 240 mg (0.74 mmol) of Cs2CO3. After completion of the reaction, monitored by TLC (DCM), the mixture was filtered, and DMF was evaporated under reduced pressure. The product was purified by column chromatography using n-hexane/DCM (3:7, v/v) as the eluent, yielding 364 mg (0.64 mmol, 95%) of 1b. 1H NMR [parts per million (ppm)]: δ 7.78, 7.63 (4H,

Rump et al.

dd, J ) 8.9 Hz, ArH), 5.30 (3H, m, COCH2O and vinyl H), 3.51 (1H, m, H-3). 13C {1H} NMR (ppm): δ 71.7 (C3), 65.7 (COCH2O), 121.6 (C-6), 132.2, 129.3 (CH Ar), 133.0, 129.1 (qC Ar), 173.6 (C-24), 191.5 (CdO ketone). 5β-Cholanic Acid 3r-Ol (4-Bromobenzoyl)methyl Ester 2b. Compound 2b was prepared with the procedure described for 1b, starting from 865 mg (2.30 mmol) of lithocholic acid 2a. The product was purified by column chromatography with MeOH/DCM (1:99, v/v) as the eluent, yielding 1.31 g (2.28 mmol, 99%) of 2b. 1H NMR (ppm): δ 7.74 and 7.59 (4H, dd, J ) 8.6 Hz, ArH), 5.26 (2H, m, COCH2O), 3.57 (1H, m, H-3). 13C {1H} NMR (ppm): δ 65.3 (COCH2O), 70.7 (C-3), 128.5, 132.3 (qC Ar), 128.8, 131.6 (CH Ar), 173.3 (C-24), 191.4 (CdO ketone). 5β-Cholanic Acid 3r,7r-Diol (4-Bromobenzoyl)methyl Ester 3b. Compound 3b was prepared with the procedure described for 1b, starting from 500 mg (1.27 mmol) of chenodeoxycholic acid. The product was purified by column chromatography with DCM f MeOH/ DCM (1:25, v/v) as the eluent, yielding 650 mg (1.10 mmol, 87%) of 3b. 1H NMR (ppm): δ 7.79, 7.67 (4H, dd, J ) 8.6 Hz, ArH), 5.28 (2H, m, COCH2O), 3.45 (1H, m, H-3). 13C {1H} NMR (ppm): δ 65.4 (COCH2O), 67.8 (C7), 71.4 (C-3), 128.6, 132.6 (qC Ar), 129.0 and 131.8 (CH Ar), 173.2 (C-24), 191.2 (CdO ketone). 3β-(Oleoyloxy)-5-cholenic Acid (4-Bromobenzoyl)methyl Ester 1c. To a solution of 200 mg (0.35 mmol) of 1b and 47 mg (0.39 mmol) of DMAP in 10 mL of pyridine, kept under a N2 atmosphere, was added dropwise 116.5 mg (0.40 mmol) of oleoyl chloride. After being stirred for 6 h, the mixture was concentrated in vacuo. The residue was dissolved in DCM and the mixture washed with 1 N HCl and brine. The organic layer was dried over MgSO4 and evaporated to dryness. The product was purified by column chromatography using n-pentane/DCM (2:5, v/v) as the eluent, yielding 170 mg (0.20 mmol, 58%) of 1c. 1H NMR (ppm): δ 7.68, 7.52 (4H, dd, J ) 8.6 Hz, ArH), 5.26 (5H, m, COCH2O and vinyl H), 4.50 (1H, m, H-3). 13C {1H} NMR (ppm): δ 14.1 (CH3-oleoyl), 65.6 (COCH2O), 73.6 (C-3), 122.4 (C-6), 129.2 132.1 (CH Ar), 129.0, 132.9 (qC Ar), 129.7, 129.9 (CHd oleoyl), 139.6 (C-5), 173.2 (CdO oleoyl ester), 173.5 (C-24), 191.4 (CdO ketone). 3r-(Oleoyloxy)-5β-cholanic Acid (4-Bromobenzoyl)methyl Ester 2c. Compound 2c was prepared with the procedure described for 1c, starting from 470 mg (0.82 mmol) of 2b. The product was purified by column chromatography using n-pentane/DCM (2:3, v/v) as the eluent, yielding 650 mg (0.78 mmol, 95%) of 2c. 1H NMR (ppm): δ 7.78 and 7.63 (4H, dd, J ) 8.2 Hz, ArH), 5.34 (4H, m, COCH2O and vinyl H), 4.75 (1H, m, H-3). 13C {1H} NMR (ppm): δ 14.0 (CH3-oleoyl), 65.6 (COCH2O), 73.9 (C-3), 129.2, 132.1 (CH Ar), 129.0, 132.9 (qC Ar), 129.7, 129.9 (CHd oleoyl), 173.2 (CdO oleoyl ester), 173.5 (C-24), 191.3 (CdO ketone). 3r,7r-Bis(oleoyloxy)-5β-cholanic Acid (4-Bromobenzoyl)methyl Ester 3c. To a solution of 340 mg (0.58 mmol) of 3b in 10 mL of pyridine were added 155 mg (1.3 mmol) of DMAP and 451 mg (1.5 mmol) of oleoyl chloride. After the mixture was stirred for 2 h, another portion of 100 mg of DMAP and 226 mg of oleoyl chloride were added. After being stirred overnight, the mixture was concentrated in vacuo. The residue was dissolved in DCM, and the organic layer was washed with 1 N HCl and brine. The organic layer was dried over MgSO4. The product was purified by column chromatography using DCM as the eluent, yielding 150 mg (0.13 mmol, 23%) of the dioleate 3c and 210 mg (0.27 mmol, 46%) of the monooleate (C-3-esterified). 1H NMR (ppm): δ 7.78 and

Synthesis of Lipidic ODNs and Their Binding to LDL

7.62 (4H, dd, J ) 8.9 Hz, ArH), 5.34 (6H, m, COCH2O and vinyl H), 4.89 (1H, m, H-7), 4.60 (1H, m, H-3). 13C {1H} NMR (ppm): δ 14.0 (CH3-oleoyl), 65.6 (COCH2O), 70.8 (C-7), 73.7 (C-3), 129.1, 132.1 (CH Ar), 129.0, 132.9 (qC Ar), 129.1, 129.9 (CHd oleoyl), 172.9, 173.2, 173.4 (2× CdO oleoyl ester, C24). 3β-(Oleoyloxy)-5-cholenic Acid 1d. To a solution of 170 mg (0.20 mmol) of 1c in 20 mL of DCM/HAc (1:1, v/v) was added 100 mg of Zn (powder). After being stirred overnight, the mixture was filtered over Hyflo and concentrated in vacuo. The product was purified by column chromatography using MeOH/DCM (1:99, v/v) as the eluent, yielding 124 mg (0.19 mmol, 97%) of 1d. 1H NMR (ppm): δ 5.32 (3H, m, vinyl H), 4.60 (1H, m, H-3). 13C {1H} NMR (ppm): δ 14.1 (CH -oleoyl), 73.6 (C-3), 3 122.4 (C-6), 129.7, 129.9 (CHd oleoyl), 139.6 (C-5), 173.3 (CdO oleoyl ester), 180.3 (C-24). MS(FAB): 637.5 (M H). HRMS: Calcd for C42H69O4 637.5196, found 637.5189. 3r-(Oleoyloxy)-5β-cholanic Acid 2d. Compound 2d was prepared with the procedure described for 1d, starting from 650 mg (0.78 mmol) of 2c. The product was purified by column chromatography using MeOH/DCM (from 1:99 to 1:66, v/v) as the eluent, yielding 460 mg (0.72 mmol, 92%) of 2d. 1H NMR (ppm): δ 11.06 (1H, m, COOH), 5.33 (2H, m, vinyl H), 4.72 (1H, m, H-3). 13C {1H} NMR (ppm): δ 14.0 (CH3-oleoyl), 74.0 (C-3), 129.7, 129.9 (CHd oleoyl), 173.4 (CdO oleoyl ester), 180.5 (C24). MS(FAB): 639 (M - H). HRMS: Calcd for C42H71O4 639.5352, found 639.5399. 3r,7r-Bis(oleoyloxy)-5β-cholanic Acid 3d. Compound 3d was prepared with the procedure described for 1d, starting from 150 mg (0.13 mmol) of 3c. The product was purified by column chromatography using MeOH/ DCM (from 1:99 to 2:99, v/v) as the eluent, yielding 96 mg (0.10 mmol, 80%) of 3d. 1H NMR (ppm): δ 5.34 (4H, m, vinyl H), 4.89 (1H, m, H-7), 4.59 (1H, m, H-3). 13C {1H} NMR (ppm): δ 13.9 (CH3-oleoyl), 70.8 (C-7), 73.7 (C-3), 129.5, 129.8 (CHd oleoyl), 172.8, 173.1 (2× CdO oleoyl ester), 179.5 (C-24). MS(FAB): 920 (M - H). HRMS: Calcd for C60H103O6 919.7755, found 919.7819. 3β-(Oleoyloxy)-5-cholenic Acid Pentafluorophenyl Ester 1e. To a solution of 90 mg (0.14 mmol) of 1d in 10 mL of DCE was added 30 mg (0.16 mmol) of pentafluorophenol, and the mixture was cooled to 0 °C. Subsequently, 35 mg (0.17 mmol) of DCC was added. After 0.5 h, the mixture was allowed to warm to room temperature. After precipitation of dicyclohexylurea, the mixture was filtered and concentrated in vacuo. The product was purified by column chromatography using hexane/DCM (11:9, v/v) as the eluent, yielding 110 mg (0.14 mmol, 98%) of 1e as a colorless oil. 1H NMR (ppm): δ 5.34 (3H, m, vinyl H), 4.59 (1H, m, H-3), 0.88 (3H, t, CH3-oleoyl). 13C {1H} NMR (ppm): δ 73.6 (C-3), 122.4 (C-6), 129.7, 129.9 (CHd oleoyl), 139.7 (C-5), 169.9 (C-24), 173.2 (CdO oleoyl ester). 19F NMR (ppm): δ -151.1 (m), -156.5 (t), -160.7 (m). 3r-(Oleoyloxy)-5β-cholanic Acid Pentafluorophenyl Ester 2e. Compound 2e was prepared with the procedure described for 1e, starting from 260 mg (0.41 mmol) of 2d. The product was purified by column chromatography with hexane/DCM (2:3, v/v) as the eluent, yielding 325 mg (0.40 mmol, 98%) of 2e as a colorless oil. 1H NMR (ppm): δ 5.34 (2H, m, vinyl H), 4.73 (1H, m), 0.88 (3H, t, CH3-oleoyl). 13C {1H} NMR (ppm): δ 14.1 (CH3-oleoyl), 74.0 (C-3), 129.7, 130.0 (CHd oleoyl), 169.9 (C-24), 173.3 (CdO oleoyl ester). 19F NMR (ppm): δ -151.1 (m), -156.5 (t), -160.7 (m). 3r,7r-Bis(oleoyloxy)-5β-cholanic Acid Pentafluorophenyl Ester 3e. Compound 3e was prepared with

Bioconjugate Chem., Vol. 9, No. 3, 1998 343

the procedure described for 1e, starting from 96 mg (0.10 mmol) of 3d. The product was purified by column chromatography with hexane/DCM (1:3, v/v) as the eluent, yielding 77 mg (0.07 mmol, 68%) of 3e as a colorless oil. 1H NMR (ppm): δ 5.33 (4H, m, vinyl H), 4.90 (1H, m, H-7), 4.60 (1H, m, H-3), 0.88 (3H, t, CH3oleoyl). 13C {1H} NMR (ppm): δ 14.0 (CH3-oleoyl), 70.8 (C-7), 73.7 (C-3), 129.6, 129.9 (CHd oleoyl), 169.8 (C-24), 172.9, 173.1 (2× CdO oleoyl ester). 19F NMR (ppm): δ -151.1 (m), -156.5 (t), -160.7 (m). 5β-Cholanic Acid 3r-Ol Pentafluorophenyl Ester 2f. To a solution of 150 mg (0.40 mmol) of lithocholic acid 2a in 5 mL of DCE/DMF (4:1, v/v) was added 88 mg (0.44 mmol) of pentafluorophenol, and the mixture was cooled to 0 °C. Subsequently, 99 mg (0.44 mmol) of DCC was added. After 0.5 h, the mixture was allowed to warm to room temperature and stirring was continued until dicyclohexylurea precipitated. The mixture was filtered and concentrated in vacuo. The product was purified by column chromatography using DCM/MeOH (98:2, v/v) as the eluent, yielding 194 mg (0.36 mmol, 89%) of 2f as a white solid. 1H NMR (ppm): δ 3.73 (1H, m, H-3). 13C {1H} NMR (ppm): δ 73.6 (C-3), 122.4 (C-6), 129.7, 129.9 (CHd oleoyl), 139.7 (C-5), 169.9 (C-24), 173.2 (CdO oleoyl ester). 19F NMR (ppm): δ -151.1 (m), -156.6 (t), -160.7 (m). Oleic Acid Pentafluorophenyl Ester 4b. Compound 4b was prepared with the procedure described for 2f, starting from 200 mg (0.71 mmol) of oleic acid 4a. The product was purified by column chromatography with n-hexane/DCM (9:1, v/v) as the eluent, yielding 230 mg (0.51 mmol, 72%) of 4b as a colorless oil. 1H NMR (ppm): δ 5.35 (2H, m, vinyl H), 0.88 (3H, t, CH3-oleoyl). 13 C {1H} NMR (ppm): δ 14.04 (CH3-oleyl), 129.63 and 130.06 (CHd oleyl), 169.52 (CdO). 19F NMR (ppm): δ -151.1 (m), -156.6 (t), -160.7 (m). Radiolabeling of ODN by Hydrogen-Tritium Exchange. The 3′-amine ODN was tritiated by the procedure of Graham et al. (27), with slight modifications. After 0.50 mg of the 3′-amine ODN was dissolved in 200 µL of 50 mM sodium phosphate buffer (pH 7.8), containing 0.1 mM EDTA, the solution was freeze-dried. The lyophilized material was dissolved in 200 µL of 3H2O (0.6 Ci), and 4.2 µL of β-mercaptoethanol was added. The solution was covered with 400 µL of mineral oil and was incubated in a gasketed vial in an oil bath kept at 90 °C. After 6 h, the radiolabeled ODN was desalted and depleted of 3H2O by repetitive centrifugation and resuspension in 1H2O using a Microcon-3 concentrator (Amicon, Witten, Germany). To remove final traces of 3H2O, the concentrated [3H]ODN was taken up in 50 µL of H2O and precipitated with 500 µL of 3% LiClO4 in acetone. Finally, the precipitate was taken up in H2O, and after the A260 and radioactivity were measured, the ODN was stored at -20 °C. We did not detect any significant exchange of radioactivity upon 6 months of storage at -20 °C. The total yield was 0.28 mg (56%). The specific activity of the [3H]ODN was 80 × 106 dpm mg-1 (0.5 × 109 dpm µmol-1). Synthesis and Purification of 3′-Lipid-ODN. The 3′-amine ODN was precipitated as a Li salt with 10 volume equiv of 3% LiClO4 in acetone. Next, the ODN was dissolved in H2O and precipitated again with 10 volume equiv of acetone to remove final traces of LiClO4. In a typical derivatization experiment, 15 nmol of ODN was dissolved in 350 µL of H2O/DMF/dioxane (1:4:2, v/v/ v). Subsequently, 1 µmol of the activated lipid dissolved in 100 µL of dioxane and 35 µmol of DiPEA were added. The mixture was incubated for 48 h at 56 °C for lipids

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1e, 2e, and 3e or at 37 °C for 2f, 4b, and cholesteryl chloroformate. Solvents were removed in a speed-vac concentrator, and the residue was taken up in 200 µL of DCM and 200 µL of H2O. The layers were separated by centrifugation, and the organic layer was washed twice with 200 µL of H2O. The H2O fractions were combined and freeze-dried. Derivatization of [3H]ODN was performed as described above with 2 nmol of [3H]ODN and 4 nmol of unlabeled ODN. ODNs conjugated with lithocholic acid, oleic acid, or cholesterol were purified by reversed phase (RP)-HPLC on a Waters C4 column (5 µm, 300 Å, 300 × 3.9 mm), applying a gradient of 1% CH3CN/min in 50 mM triethylammonium acetate (pH 7.0) at a flow rate of 1 mL/min. The gradient (5 to 50%) was started after elution for 5 min at 5% CH3CN. ODNs were detected at 260 nm. All other lipid-conjugated ODNs were purified by gel electrophoresis in a 1% LM-MP agarose gel containing 0.1% Tween 20, in 0.5× TBE [45 mM Tris-borate and 0.1 mM EDTA (pH 8.4)]. After separation of the lipid-ODN from the nonconjugated ODN, the gel slice containing the lipid-ODN was melted for 5 min at 65 °C. The agarose was digested with Agarase (40 units per milliliter of gel) at 45 °C in 30 mM Bis-Tris and 10 mM EDTA for 2 h. The lipid-ODNs were precipitated with 10 volume equiv of acetone. To remove traces of undigested agarose, the precipitate was taken up in 200 µL of H2O and passed over a filter paper (no. 589, Schleicher and Schu¨ll). Lipidconjugated ODNs were isolated in a yield of 35-75%. Determination of Melting Temperatures. Melting temperatures of hybrids of the antisense 3′-lipid-ODNs with an 28-mer sense ODN were determined using a Perkin-Elmer spectrophotometer equipped with a PTP-6 thermal programmer. Equimolar amounts of both ODNs were dissolved in PBS (pH 7.4) to a concentration of 3.6 µM, denatured at 96 °C for 2 min, and subsequently cooled to room temperature to anneal slowly. Then, the temperature was adjusted to 35 °C, and hybrids were melted by increasing the temperature to 95 °C at a rate of 0.5 °C min-1. Duplicate runs produced Tms within 1 °C. Isolation and Radiolabeling of LDL. LDL was isolated from the serum of fasted volunteers by density gradient ultracentrifugation (28) and was dialyzed against PBS containing 1 mM EDTA (pH 7.4). Radioiodination was performed at pH 10.0 with carrier-free 125I as described by McFarlane (29). Protein concentrations of LDL were determined by the method of Lowry (30) with bovine serum albumin as a standard. Determination of the Association of Lipid-ODNs with LDL. Equimolar amounts of [125I]LDL and lipid[3H]ODNs (1.7 µM), dissolved in PBS and 1 mM EDTA (pH 7.4), were incubated for 2 h at 37 °C. For these experiments, the molecular weight of an LDL particle was related to the molecular weight of the apoprotein B-100 (31), as each LDL particle contains only one copy of the apoprotein. The molecular weights of the lipidODNs were taken from Table 1. Aliquots of the incubation mixtures were subjected to gel electrophoresis in a 0.75% (w/v) agarose gel in 75 mM Tris-hippuric acid buffer (pH 8.8). After electrophoresis, the gel was cut into slices, and after addition of 0.5 mL of Soluene-350, the 125I radioactivity was counted. After 24 h, 3 mL of Hionic Fluor was added and samples were evaluated for 3H radioactivity. The measured values of 3H radioactivity were corrected for the contribution of 125I radioactivity.

Rump et al. Table 1. Synthesis and Analysis of Lipid-ODNs

a Melting temperatures (degrees Celsius) were calculated from duplicate runs with a 28-mer unmodified DNA strand as the target sequence in PBS. b Lipid-ODNs were analyzed by ES mass spectrometry. The exact mass data were calculated from at least four multiply charged ions. c Cholesteryl chloroformate.

RESULTS AND DISCUSSION

Synthesis of Activated Lipid Structures. Starting points for the design of the lipids to be conjugated to the c-myb specific ODNs were the studies of Firestone et al. (13, 14). These studies show that aliphatic lipids with at least one cis double bond and oleoyl steroid esters facilitate reconstitution of LDL with lysosomotropic agents and nitrogen mustards. The lipid structures in this study were synthesized, as shown in Scheme 1, starting from oleic acid 4a and the bile acids cholenic acid 1a, lithocholic acid 2a, and chenodeoxycholic acid 3a. Bile acids were selected because they contain both a carboxylic acid functionality which can be used for conjugation to ODNs bearing nucleophilic moieties and hydroxyl groups that can be esterified with fatty acids. The lipophilicity of the bile acids was enhanced by introducing one oleic acid chain (1d and 2d) or two oleic acid chains (3d). The bile acids lithocholic acid 2a and cholenic acid 1a differ in the configuration of the hydroxyl at the 3 position and the rigidity of the steroid rings. First, the carboxylic acids of the bile acids were protected with 4-bromophenacyl bromide. Then, the hydroxyls were esterified with oleoyl chloride in the presence of DMAP, resulting in the oleoyl steroid esters 1c and 2c in a yield of 58-95%. Synthesis of 3c afforded the dioleoyl ester in a low yield (23%), compared to those of the monooleoyl esters 1c and 2c. Characterization of the reaction products formed during synthesis of 3c revealed two main products: the dioleoyl ester 3c and the monooleoyl steroid ester esteri-

Synthesis of Lipidic ODNs and Their Binding to LDL

Bioconjugate Chem., Vol. 9, No. 3, 1998 345

Scheme 1. Synthesis of Activated Lipid Structuresa

fied at the 3-OH. The 7-OH monooleoyl ester could not be detected as a main product. The 3-OH of chenodeoxycholic acid thus appears to react more easily with oleoyl chloride than the 7-OH, presumably because this position is more readily accessible. Upon formation of the oleic acid ester at the 3-OH, the 7-OH may become even less accessible for esterification, due to steric hindrance. We also attempted to synthesize the dioleoyl steroid ester of deoxycholic acid (3-OH and 12-OH). However, compared to the 7-OH of chenodeoxycholic acid, the 12-OH of deoxycholic acid was found to be even more difficult to esterify. After esterification of the hydroxyls of the steroids, the bromophenacyl group could easily be removed by overnight stirring in HAc/Zn. All synthesized lipids were activated with pentafluorophenol to allow coupling to 3′amino-tailed ODNs. Conjugation of ODNs with Lipid Structures. We performed synthesis of the lipid-ODNs in solution phase, since in the alternative approach, solid phase synthesis, the steroid ester structures would be disrupted by the standard ammonia cleavage of the ODN from the solid support (32, 33). However, earlier studies have proven that the covalent attachment of large lipids to ODNs in solution phase is a difficult task. The lipids and the ODNs have very different solubility properties, and the amphiphilic properties of the resulting lipid-ODNs often lead to isolation problems and consequently low yields (34-36). To achieve a high conjugation efficiency for our

lipids, we have tested several protocols described in the literature. Derivatization of ODNs with lipids in aqueous buffer solutions (pH 8-10) has been reported to be the most successful derivatization method (32, 33). However, the low solubility of most of our lipids in these mixtures resulted in very low yields of the conjugated ODN. Lipophilic cations such as CTAB or PMDBD have been used to dissolve and subsequently derivatize ODNs with lipids in organic solvents such as DMSO and CHCl3 (34, 35, 37, 38). Unfortunately, this approach failed for derivatization of our lipids. We found that derivatization in a mixture of H2O and the aprotic solvents DMF and 1,4-dioxane (1:4:4, v/v/v), in which DiPEA was present to create mildly basic conditions, is far superior to the other two methods (39). The presence of dioxane in this mixture improved the solubility of the steroid lipids, and DMF appeared to be a crucial component. When DMF was replaced by dioxane, resulting in a mixture of H2O and dioxane only (1:8, v/v), the conjugations were unsuccessful, although both the ODN and the activated lipid could be dissolved. Derivatization of the 3′-amino ODN could efficiently be accomplished in the H2O/DMF/dioxane mixture for 48 h at 37 °C for the lipids 2f, 4b, and cholesteryl chloroformate. The activated lipids 1e, 2e, and 3e were conjugated in the same mixture, but the temperature was raised to 56 °C to ensure complete solubility of these activated steroid lipids. After conjugation of the ODN and removal of the reaction mixture solvents, excess reactive lipids were

346 Bioconjugate Chem., Vol. 9, No. 3, 1998

extracted with DCM. Without this extraction step, recoveries of the conjugated ODNs were very low. Separation of the ODNs conjugated with lithocholic acid, oleic acid, and cholesterol (ODN-2, ODN-3, and ODN-4, respectively) from the nonconjugated ODNs was accomplished by RP-HPLC (Waters C4 column, 5 µm, 300 Å) using an CH3CN gradient from 5 to 50% in 50 mM triethylammonium acetate (pH 7.0). The retention time of ODNs increased significantly from 17 min for the nonconjugated ODN to 23.7 min for ODN-2 and 34.5 min for ODN-3 and ODN-4. When a C8 HPLC column was utilized, the cholesteryl-derivatized ODN had a much higher retention time than the oleic acid-derivatized ODN. The C4 column was preferred since it led to higher recoveries of the ODNs (overall recoveries of up to 80%). Purification of the ODNs conjugated with the oleoyl steroid ester structures (ODN-5, ODN-6, and ODN-7) was not possible by RP-HPLC; no conjugated products could be recovered. Several RP-HPLC columns (C4, C8, and C18) were tested, and very high concentrations of the organic modifier (CH3CN and tetrahydrofuran up to 95%) were used. Only small amounts (95%.

of lithocholic acid and cholenic acid associate spontaneously and almost completely with LDL. More than 95% of the [3H]ODNs conjugated with these steroids comigrated in the gel with radioiodinated LDL at an Rf of 0.2. These steroids meet the structural requirements for LDL anchors as defined by Firestone (13). Association of the lipid-ODNs with LDL also slightly broadened the band of LDL in the gel. This is probably due to an increase of the overall negative charge of the complex. When ODNs were associated at higher molar ratios, the increase in the electrophoretic mobility was more clear (data not shown). The association of cholesteryl-derivatized ODNs with native LDL has been shown before (10, 19), but the use of oleoyl steroid esters to induce association of ODNs with LDL is novel. In Figure 2, the fact that not all lipids can induce spontaneous association of the ODN with LDL is also illustrated. Lithocholic acid and oleic acid proved not to be effective as a LDL anchors in our experiments. This also applies for the bis-oleoyl steroid ester. Only a small proportion of the ODN conjugated to this steroid ester (ODN-6) comigrated with LDL, and more than 50% of the 3H radioactivity was found at an Rf of >0.2. The aberrant migration pattern for ODN-6 may indicate that this lipid-ODN has formed an aggregate during incubation with LDL or that it slowly dissociates from the LDL during electrophoresis. The lipid moiety of ODN-6 probably does not partition with its complete steroid structure into the lipids of LDL, but only with the two oleoyl chains. The complete bis-oleoyl steroid ester structure may be too bulky to associate spontaneously with LDL. How-

ever, this ODN might be an interesting candidate to use in the recently developed artificial LDL-like carrier systems (43, 44). In this system, lipid-ODNs are incorporated during vesicle formation, in contrast to the spontaneous association with LDL in this study. The lack of association of ODN-3 with LDL was not expected, since complexation of a 5′-palmitoylated 16-mer phosphorothioate ODN with LDL was already reported by Mishra et al. (20). The structural difference between oleic acid and palmitic acid may explain the difference in LDL association. In fact, Svinarchuk et al. (8) reported that small structural differences of fatty acids can change their binding characteristics for membrane structures significantly. Alternatively, the inability of the oleoyl chain to associate ODNs with LDL may be caused by different methods of analysis of complexation. Concluding Remarks. We have efficiently conjugated ODNs to several lipid structures in solution phase. The conjugates could easily be purified in high yields, either by RP-HPLC or by electrophoresis in a 1% agarose gel in the presence of 0.1% detergent. Several of the lipid-ODN conjugates associate readily with LDL, which opens the possibility of utilizing the lipid structures for specific delivery of oncogene-directed ODNs to tumor cells via the LDLr pathway. To be effective, however, the ODNs need to escape from the endosomal/lysosomal compartment. We speculate that lipid-ODNs can exit these compartments, since it has been shown by several authors that lipid-ODNs can bind to membrane structures which may facilitate transport. Indeed, enhanced activity of lipid-ODNs, which may be the result of

348 Bioconjugate Chem., Vol. 9, No. 3, 1998

increased transport across the cellular membrane, has been described (7, 9). ACKNOWLEDGMENT

Muthiah Manoharan and Kathleen Tivel at ISIS Pharmaceuticals (Carlsbad, CA) and Sven Wagner at the DKFZ (Heidelberg, Germany) are acknowledged for mass analyses of the ODNs. Dr. S. van Zwanenbergstichting is acknowledged for financial support for presenting part of this work at the 12th IRT (1996, La Jolla, CA). LITERATURE CITED (1) Wagner, R. W., and Flanagan, W. M. (1997) Antisense technology and prospects for therapy of viral infections and cancer. Mol. Med. Today 3 (1), 31-38. (2) Szymkowski, D. E. (1996) Developing antisense oligonucleotides from the laboratory to clinical trials. Drug Discovery Today 1 (10), 415-428. (3) Boutorine, A. S., and Kostina, E. V. (1993) Reversible Covalent Attachment of Cholesterol to Oligodeoxyribonucleotides for Studies of the Mechanisms of Their Penetration into Eucaryotic Cells. Biochimie 75 (1-2), 35-41. (4) Boutorine, A. S., Gus’kova, L. V., Ivanova, E. M., Kobetz, N. D., Zarytova, V. F., Ryte, A. S., Yurchenko, L. V., and Vlassov, V. V. (1989) Synthesis of alkylating oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3′-terminus and their interaction with DNA within mammalian cells. FEBS Lett. 254, 129-132. (5) Vu, H., Singh, P., Lewis, L., Zendegui, J. G., and Jayaraman, K. (1993) Synthesis of cholesteryl supports and phosphoramidate for automated DNA synthesis of triple-helix forming oligonucleotides (TFOs). Nucleosides Nucleotides 12, 853-864. (6) Shea, R. G., Marsters, J. C., and Bischofberger, N. (1990) Synthesis, hybridization properties and antiviral activity of lipid-oligonucleotide conjugates. Nucleic Acids Res. 18, 37773783. (7) Alahari, S. K., Dean, N. M., Fisher, M. H., Delong, R., Manoharan, M., Tivel, K. L., and Juliano, R. L. (1996) Inhibition of expression of the multidrug resistance-associated P-glycoprotein by phosphorothioate and 5′ cholesterolconjugated phosphorothioate antisense oligonucleotides. Mol. Pharmacol. 50 (4), 808-819. (8) Svinarchuk, F. P., Konevetz, D. A., Pliasunova, O. A., Pokrovsky, A. G., and Vlassov, V. V. (1993) Inhibition of HIV Proliferation in MT-4 Cells by Antisense Oligonucleotide Conjugated to Lipophilic Groups. Biochimie 75 (1-2), 4954. (9) Desjardins, J., Mata, J., Brown, T., Graham, D., Zon, G., and Iversen, P. (1995) Cholesteryl-conjugated phosphorothioate oligodeoxynucleotides modulate CYP2B1 expression in vivo. J. Drug Targeting 2 (6), 477-485. (10) Krieg, A. M., Tonkinson, J., Matson, S., Zhao, Q., Saxon, M., Zhang, L.-M., Bhanja, U., Yakubov, L., and Stein, C. A. (1993) Modification of antisense phosphodiester oligodeoxynucleotides by a 5′ cholesteryl moiety increases cellular association and improves efficacy. Proc. Natl. Acad. Sci. U.S.A. 90, 1048-1052. (11) Farooqui, F., Sarin, P. S., Sun, D., and Letsinger, R. L. (1991) Effect of structural variations in cholesteryl-conjugated oligonucleotides in inhibitory activity toward HIV-1. Bioconjugate Chem. 2, 422-426. (12) Brown, M. S., and Goldstein, J. L. (1986) A receptormediated pathway for cholesterol homeostasis. Science 232, 34-47. (13) Firestone, R. A., Pisano, J. M., Falck, J. R., McPaul, M. M., and Krieger, M. (1984) Selective delivery of cytotoxic compounds to cells by the LDL pathway. J. Med. Chem. 27, 1037-1043. (14) Dubowchik, G. M., and Firestone, R. A. (1995) Improved cytotoxicity of antitumor compounds deliverable by the LDL pathway. Bioconjugate Chem. 6, 427-439. (15) Firestone, R. A. (1994) Low-density lipoprotein as a vehicle for targeting antitumor compounds to cancer cells. Bioconjugate Chem. 5 (2), 105-113.

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