Novel Intracellular Delivery System of Antisense Oligonucleotide by

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Bioconjugate Chem. 2003, 14, 473−479

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Novel Intracellular Delivery System of Antisense Oligonucleotide by Self-Assembled Hybrid Micelles Composed of DNA/PEG Conjugate and Cationic Fusogenic Peptide Ji Hoon Jeong,† Sung Wan Kim,‡ and Tae Gwan Park*,† Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, South Korea and CCCD/Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112. Received October 27, 2002; Revised Manuscript Received January 13, 2003

An antisense oligonucleotide (ODN), c-myb, was covalently conjugated to poly(ethylene glycol) (PEG) via an acid-cleavable phosphoramidate linkage to form a diblock copolymer-like structure. The phosphoramidate linkage between ODN and PEG was completely cleaved within 5 h in an endosomal acidic condition (pH 4.7). When complexed with a cationic fusogenic peptide, KALA, the ODN/PEG conjugate self-associated to form polyelectrolyte complex micelles in an aqueous solution. The anionic ODN segments were ionically interacted with cationic KALA peptide to form an inner polyelectrolyte complex core, while the PEG segments constituted a surrounding corona. Effective hydrodynamic volume of the micelles was ca. 70 nm with a very narrow size distribution. The polyelectrolyte complex micelles, composed of c-myb ODN-PEG conjugate and KALA, were transported into cells far more efficiently than c-myb ODN itself. They also exhibited higher antiproliferative activity against smooth muscle cells. This study demonstrates that the DNA/PEG hybrid micelles system can be applied for the delivery of antisense oligonucleotide.

INTRODUCTION

A wide variety of antisense oligonucleotides (ODN) have drawn much attention due to their specific interaction with cytoplasmic mRNA to block specific gene expression (1-4). Modulating the expression of a target gene by ODN has considerable potential for diverse therapeutic applications (5-8). Practical applications of antisense ODN as therapeutic chemical entities, however, have two inherent problems: susceptibility to enzymatic hydrolysis and poor cellular uptake (9, 10). The enzyme stability problem can be partly improved by synthesizing chemically modified ODN derivatives such as phosphorothioates and peptide nucleic acids. The modified ODN derivatives showed resistant to the enzymatic digestion by nucleases (11, 12). The limited cellular uptake of antisense ODN has yet been fully solved. Since ODN is a negatively charged macromolecular drug having a very low permeability across cell membrane, high dose of ODN is generally required to achieve the desired antisense effect. To increase the cellular uptake of ODN, antisense ODN was formulated with liposomes (13, 14), cationic lipids (15), and cationic polymers (16). These formulations are based on the formation of polyelectrolyte complex nanoparticles that can be more readily taken up by cells via an endocytosis pathway rather than a passive diffusion route. Significantly improved extent of cellular uptake as well as enhanced stability of ODN can be achieved using these nanoscale and nonviral vector formulations. We recently reported a new DNA/polymer hybrid micelle structure as an ODN carrier. An antisense ODN * Corresponding author. Tel: +82-42-869-2621, fax: +82-42869-2610, e-mail: [email protected]. † Korea Advanced Institute of Science and Technology. ‡ University of Utah.

was covalently conjugated to the terminal end of a biodegradable polymer, poly(D,L-lactic-co-glycolic acid) (PLGA). The ODN/PLGA conjugate has an amphiphatic diblock copolymer structure composed of a hydrophilic ODN segment and a hydrophobic PLGA segment. The ODN/PLGA conjugate self-assembled to form micelles in an aqueous medium. The PLGA segment in the conjugate constituted an inner core while the ODN segment, highly hydrophilic and ionized, formed a surrounding corona (10). These ODN/PLGA micelles could be more efficiently transported within cells, suggesting that the micelle-type carriers could be an efficient alternative carrier for ODN delivery. In the present study, antisense ODN, c-myb, was chemically conjugated with PEG via an acid cleavable linkage to produce an ODN-PEG conjugate. It was hypothesized that the ODN-PEG conjugate could selfassemble to form polyelectrolyte complex micelles by formulating with various cationic peptides and polymers. Ionic interaction between anionic ODN segments and cationic counterparts was expected to drive the formation of a polyeletrolyte complex inner core, whereas PEG segments constitute a surrounding corona. The entrapment of nucleic acids in the core of polyelectrolyte complex micelles could significantly improve the nuclease resistance of antisense ODN from enzymatic attack (1719). The surrounding hydrophilic PEG segments not only improve the solubility of the nanoparticulate in aqueous milieu (20, 21), but also protect ODN from serum protein binding, owing to the steric hindrance effect of the flexible PEG chains (22-24). Physical properties of the polyelectrolyte complex micelles were characterized. Cellular uptake behaviors and antiproliferation effect of c-myb antisense ODN on smooth muscle cells were also investigated.

10.1021/bc025632k CCC: $25.00 © 2003 American Chemical Society Published on Web 03/05/2003

474 Bioconjugate Chem., Vol. 14, No. 2, 2003 MATERIALS AND METHODS

Materials. Oligonucleotide (antisense c-myb ODN, 5′-GTG-TCG-GGG-TCT-CCG-GGC-3′ and mis-matched ODN, 5′-GTC-TCC-GGC-TCA-CCC-GGG-3′), which has a terminal phosphate group at its 5′ end, was synthesized and purified by Bioneer (Taejon, Korea). Cationic peptide, KALA (WEAKLAKALAKALAKHLAKA LAKALKACEA) was synthesized and purified by Peptron (Taejon, Korea). 1-Ethyl-3,3-dimethylaminopropylcarbodiimide (EDC), imidazole, ethylenediamine, protamine, and (3-(4,5-dimethylthyazolyl-2)-2,5-diphenyltetrazolium bromide) (MTT) were purchased from Sigma (St. Louis, MO). N-Hydroxylsuccinimide-derivatized poly(ethylene glycol) (SPA-PEG, MW 2000) was obtained from Shearwater (Huntsville, AL). Sephadex G-50 resin was purchased from Sigma (St. Louis, MO). Polyethylenimine (branched PEI, MW 25000) was purchased from Aldrich (Milwaukee, WI). Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s modified phosphate-buffered saline (DPBS), and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY). All other chemicals were of analytical grade. Synthesis of ODN-PEG Conjugate. To form an active ODN-phosphoimidazolide intermediate, ODN (1 mg, 180 nmol) and EDC (3.5 mg, 18 µmol) were dissolved in 0.5 mL of 0.1 M imidazole (pH 6.0). The reaction mixture was incubated for 1 h at room temperature. The ODN having an imidazolide group at its 5′ position was isolated from the mixture by Sephadex G-50 spin-column chromatography and collected in 100 µL of 10 mM phosphate buffer containing 100 mM NaCl and 1 mM EDTA, pH 7.5. One hundred microliters of 0.5 M ethylenediamine (pH 7.7) was added to the solution containing 5′-imidazolide ODN. The reaction was carried out for 1 h at 50 °C. The ODN was then isolated by Sephadex G-50 spun-column chromatography. The conjugation of 5′ethylenediamine-ODN to PEG was performed by adding NHS-derivatized PEG. The stoichiometric molar ratio (ODN/PEG) of the conjugation reaction was 1:5. The reaction mixture was incubated for 3 h at room temperature and dialyzed against deionized water to remove unreacted PEG (MWCO 3500). Reversed-Phase Chromatography Analysis. The product from each reaction step was analyzed by an HPLC system (Waters 486, Waters, Milford, MA) equipped with UV detector (UV486, Waters, Milford, MA). The chromatography was performed on Hipore RP304 column (250 × 4.6 mm, Biorad, Hercules, CA) with detection at 260 nm using a linear gradient elution of 100 mM ammonium acetate/acetonitrile from 5/95 (v/v) to 50/50 (v/v) with a flow rate of 1 mL/min. The eluate was monitored by UV absorption measurement at 260 nm. To observe the acid-cleavage profile of ODN-PEG conjugate, the samples were incubated at either pH 4.7 (10 mM citric acid/sodium citrate buffer, pH 4.7) or pH 7.4 (10 mM phosphate buffer, pH 7.4). The samples were incubated at 37 °C. After 5 h, the sample incubated in pH 4.7 was neutralized by adding 0.1 N NaOH and then injected to the HPLC system for analysis. Free ODN was also analyzed in the same HPLC system to identify its retention time. Formation and Characterization of Polyelectrolyte Complex Micelles. Desired amounts of ODN-PEG conjugate and KALA were separately diluted in PBS and filtered through a 0.2 µm filter (Millipore, Bedford, MA). The diluted KALA solution was added to the ODN-PEG conjugate solution and mixed to form polyelectrolyte complex micelles, in which the stoichiometric molar ratio between the number of phosphate in ODN and the

Jeong et al.

number of lysine residues in KALA was adjusted at 1:1.57. The micelles were left for 30 min prior to use. Hydrodynamic volume and surface charge of the polyelectrolyte complex micelles were measured by dynamic light scattering at 25 °C using a dynamic light scattering photometer (Zeta Plus, Brookhaven Instrument Co., NY) equipped with He-Ne laser at a wavelength of 632 nm. Cell Culture. A murine smooth muscle cell line, A7R5, was purchased from American Type Cell Culture (ATCC, Rockville, MD) and was maintained in DMEM supplemented with 10% FBS, streptomycin at 100 µg/mL, penicillin at 100 IU/mL, and 2 mM L-glutamine at 37 °C in a humidified 5% CO2 atmosphere. Fluorescence Labeling of ODN-PEG Conjugate. A FITC-labeled ODN having a complementary sequence to antisense c-myb ODN (sense c-myb ODN) was hybridized with either antisense c-myb ODN or antisense c-myb ODN-PEG conjugate. The mixture was then heated at 65 °C for 5 min and cooled in ice. Confocal Microscopy. Confluent cells were trypsinized and 10000 cells per well were plated in a six-well plate (35 mm diameter, Nunc) containing collagen-coated cover-glass in 1.5 mL of DMEM with 10% FBS and incubated for 24 h before further experiments. After the replacement of the medium with fresh serum-free medium, the FITC-labeled ODN-PEG/KALA polyelectrolyte complex micelles were added to make an equivalent antisense ODN concentration of 20 µg/mL. Unconjugated antisense c-myb ODN, which was also hybridized with the FITC-labeled ODN (sense strand), was used as a control. After 3 h incubation, the medium was discarded, and the cells were extensively washed with DPBS. The cells were submerged in a fixing solution (0.2% glutaraldehyde, 0.5% formaldehyde in DPBS) and stored at 4 °C for 30 min. The cells were then washed once with PBS and visualized by an LSM 510 confocal microscope (Carl Zeiss, Germany). An argon/krypton mixed gas laser with excitation line at 495 nm was used to induce FITC fluorescence. Cell Proliferation Studies. Antiproliferative effect of c-myb antisense ODN was investigated using two types of methods, direct cell counting and MTT colorimetric assay (25-27). Cells were seeded at a density of 4000 cells per cm2 in DMEM supplemented with 10% FBS. After 24 h, the medium was replaced with a medium with 0.5% FBS. The cells were maintained in the medium containing 0.5% FBS for 48 h (day 0). The cells were then supplemented with a medium with 10% FBS alone or with indicated amount of ODN formulations. For direct cell counting, the cells were trypsinized and counted by trypan blue exclusion on hemocytometer at appropriate time point. Degree of cell proliferation was also determined by using MTT assay after additional 72 h culture. RESULTS AND DISCUSSION

Polyelectrolyte complex micelles have been reported to be formed by complexing negatively charged ODN or plasmid DNA with a block or graft copolymer consisting of a cationic polymer and poly(ethylene glycol) (PEG). Poly(L-lysine)-PEG diblock copolymer (17-20) and PEGgrafted PEI copolymer (21) were reported to form selfassembled micellar associates when complexed with plasmid DNA or ODN. Electrostatic interactions between anionic ODN molecules and cationic polymer segments are responsible for the formation of charge-neutralized inner core, while PEG segments constitute a surrounding corona. In this study, ODN was conjugated to PEG via an acid cleavable linkage, phosphoramidate. The ODN-

Intracellular Delivery of Antisense Oligonucleotide

Figure 1. A schematic illustration of the formation of polyelectrolyte complex micelles self-assembled from ODN-PEG conjugate and KALA.

PEG conjugate, having a diblock copolymer structure, has two hydrophilic segments, but is expected to form polyelectrolyte complex micelles via charge-to-charge interaction between oppositely charged species of ODN segments and externally added cationic peptides. A fusogenic cationic peptide, KALA, was used in this study as a charge-neutralizing counterpart against ODN as schematically shown in Figure 1. The covalent conjugation between oligonucleotide (ODN, antisense c-myb) having a 5′ terminal phosphate group and PEG was carried out via a three-step reaction (Figure 2). The acid-labile linkage, phosphoramidate, was introduced between ODN and PEG. Coupling agents, EDC and imidazole, were used to form an active intermediate, ODN-phosphoimidazolide, which was then reacted with ethylenediamine via a phosphoramidate linkage. The resultant ethylenediamino-ODN was then coupled with a PEG derivative. The intermediate from each reaction were analyzed by HPLC, confirming the progress of conjugation reaction (data not shown). The conversion yield of ethylenediamino-ODN was ca. 90%, which is well agreed with previously published data (28). The ODNPEG conjugate had small portion of unconjugated ODN impurities (e1%) as determined by HPLC. To enhance the antisense activity of ODN, an acidcleavable phosphoramidate linkage was introduced be-

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tween ODN and PEG. It was postulated that intracellularly delivered antisense ODN should be in a PEGcleaved form to avoid the steric hindrance effect of PEG segment, which might hinder the interaction between the ODN and a target RNA sequence. The phosphoramidate linkage was reported to be labile in acidic environment, but relatively stable at neutral pH (29, 30). Considering that ODN-PEG/KALA complex micelles were first located in an acidic endosomal compartment after the endocytic cellular uptake, the acid-labile property of the phosphoramidate bond can trigger the cleavage of ODN from the ODN-PEG conjugate in the endosome compartment. The released ODN would diffuse into the cytoplasmic region where it can hybridize with its counterpart mRNA, without steric-hindrance of conjugated PEG, to block specific gene expression. Figure 3 shows cleavage profiles of ODN from ODN-PEG conjugate at different pH values. To observe the acid-sensitive cleavage profile of the phosphoramidate linkage, the ODN-PEG conjugate was incubated at pH 4.7 and pH 7.4 as a function of time. ODN was hardly cleaved when incubated at physiological pH (pH 7.4), but it was readily cleaved at pH 4.7. The cleavage of the phosphoramidate linkage at the acidic condition was completed within 5 h. The ODN-PEG conjugates could self-assemble to form polyeletrolyte complex micelles by interacting with either cationic peptides or cationic polymers as schematically illustrated in Figure 1. Since this study aims at an efficient cellular delivery system of ODN in the form of micellar nanoparticles, ODN would be preferentially transported within cells by an endocytosis mechanism, not by a passive diffusion mechanism. In this case, rapid escape of ODN from an acidic endosomal compartment to cytoplasm region is essentially needed to avoid further chemical and enzymatic degradations of ODN in later endosome and lysosomal compartments. To facilitate the escape from endosomes, ODN-PEG conjugate was complexed with a cationic fusogenic peptide, KALA, which is known to disrupt the endosomal membrane. KALA has been widely used for enhancing the transfection efficien-

Figure 2. Synthetic scheme of ODN-PEG conjugate having an acid-cleavable linkage, phosphoramidate.

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Figure 3. Cleavage profiles of ODN-PEG conjugate at pH 4.7 (A) and pH 7.4 (B). The ODN-PEG conjugate was incubated at indicated pH and analyzed by reversed-phase HPLC as described in Materials and Methods.

Figure 4. Size distribution of ODN-PEG/KALA polyelectrolyte complex micelles as determined by dynamic light scattering.

cies of various foreign plasmid genes (23, 24). KALA itself has been used as a DNA carrier for delivering plasmid DNA into cells (30, 31). To form polyelectrolyte complex micelles, stoichiometric molar ratio of the anionic phosphate group in ODN to the cationic lysine group in KALA was adjusted at 1:1.57, since KALA also contains two glutamic acid residues, which can neutralize the same number of counterions of lysine residue. The surface charge of the complex was almost neutral as determined by light scattering method (data not shown). The polyelectrolyte complex micelles generated between ODNPEG and KALA showed a hydrodynamic diameter of ca. 74 nm with a very narrow size distribution (Figure 4). The resulting uniform size of micelles suggested that ODN segments were interacted with KALA to form an inner core of the micelles by the neutralization of the two oppositely charged polyelectrolytes. PEG segments composed of a surrounding corona play an important role in stabilizing the micellar structure, which not only prevents aggregation between the micelles, but also enhances their solubility in aqueous milieu (20, 21). In contrast, the formulation of KALA with free ODN resulted in the formation of nanoparticles with about 200 nm in diameter, of which surface charge values varies depending on the molar ratio of KALA and ODN (data not shown). It is of particular interest to note that the size distribution of ODN-PEG/KALA is very narrow, which can be attributed to the monodisperse molecular weight distributions of ODN and KALA. In contrast to synthetic polymers, ODN and KALA have fixed and

Figure 5. Confocal microscopic images of smooth muscle cells treated with (A) ODN-PEG/KALA polyelectrolyte complex micelles and (B) ODN alone. The ODN-PEG conjugate and the unmodified ODN were hybridized with FITC-labeled ODN having complementary sequence with the ODN for the visualization.

monodisperse molecular weight values. Thus, stoichiometrically balanced charge-to-charge interactions between ODN and KALA within the micellar core may occur in a well-defined fashion. Uptake of ODN-PEG/KALA polyelectrolyte complex micelles by smooth muscle cells (A7R5) was visualized under confocal microscope (Figure 5). For visualization, fluorescent dye (FITC) labeled ODN having a complementary sequence to antisense c-myb (sense strand) was hybridized with the antisense c-myb ODN-PEG conjugates before forming the complex with KALA. The FITClabeled hybrid complex (antisense-ODN-PEG/sense-

Intracellular Delivery of Antisense Oligonucleotide

Figure 6. Proliferation rates of smooth muscle cells treated with no ODN (closed circle, b), mismatched antisense ODN (MAS)-PEG/KALA micelles (open circle, O), and antisense ODN (AS)-PEG/KALA micelles (inverted triangle, 1). The concentration of the antisense ODN used in each formulation was 20 µg/ mL. The experiment was carried out in triplicate.

ODN-FITC/KALA) was showed similar size and distribution to those of ODN-PEG/KALA formulation (data not shown). It can be seen that the FITC-labeled micelles were distributed over entire cytoplasm region of smooth muscle cells (Figure 5A). In contrast, the cells treated with FITC-labeled ODN showed only negligible fluorescence in cytoplasmic region, suggesting that only small fraction of ODN should be transported within the cells most likely due to its limited passive diffusion through plasma membrane (Figure 5B). This result reveals that ODN could be delivered within cells more efficiently by forming a micellar nanoparticulate structure that was more readily taken up by cells via an endocytosis mechanism. This was also in good agreement with the previous results obtained ODN-PLGA conjugate micelles (10) and PEO-PBLA[poly(β-benzyl L-aspartate)]-FITC micelles (33). The confocal image also suggests that ODN was likely to be released from endosomal compartments. This postulation was based on the observation that there were no localized fluorescent droplets scattered in the cytoplasm area, which indicate the presence of endosomes containing ODN-PEG/KALA micelles. This was possibly due to the fusogenic action of KALA that was incorporated to form an inner core of micelles. To test the efficiency of the ODN delivery system using the polyelectrolyte complex micelles, antisense ODN directed to c-myb was selected as a therapeutic ODN. Proto-oncogene c-myb is thought to play a major role in mitogen-induced proliferation of smooth muscle cells (34). An antisense c-myb has been attempted to use for the suppression of smooth muscle cell proliferation in coronary artery after a balloon angioplasty surgery procedure (35). The antisense c-myb was also reported to inhibit the proliferation of smooth muscle cells in vitro (36) and in vivo (33). Figure 6 shows the proliferation rate of smooth muscle cells (A7R5) for mismatched antisense and antisense ODN-PEG/KALA formulations at 20 µg/ mL of the antisense ODN concentration. The polyelectrolyte complex micelles containing antisense c-myb ODN showed about 70% inhibition in proliferation of the smooth muscle cells in comparison to mock-treated cells (no ODN), whereas those containing mismatched ODN exhibited no significant inhibition of proliferation (Figure 6). The 70% inhibition of the proliferation was achieved after 5 days at the concentration of 20 µg/mL of the antisense ODN in the presence of 10% FBS. This also

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Figure 7. Dose-dependent proliferation rate of smooth muscle cells treated with antisense ODN (open), antisense ODN-PEG conjugate (hatched), antisense ODN/KALA complexes (closed), and antisense ODN-PEG/KALA complex micelles (crosshatched). Degree of cell proliferation was determined by using MTT assay after 72 h from the time of addition of the formulations. The experiment was carried out in the presence of 10% FBS.

suggests the possibility that the ODN delivery system using the ODN-PEG/KALA polyelectrolyte complex micelles could protect the ODN from digestion attack by enzymes abundant in serum. It is also noted that the significant reduction of the proliferation could be achieved at relatively lower concentration (20 µg/mL) compared to previous results using antisense c-myb (36) or c-myc (37) phosphothioate ODN, in which 50% inhibition of proliferation of smooth muscle cells could be achieved only at the concentration of higher than 90 µg/mL. Figure 7 shows inhibition of proliferation of the smooth muscle cells as a function of the antisense ODN concentration for various ODN formulations. The relative proliferation rate of the cells was determined after 72 h incubation with the formulations using MTT assay and calculated as a percent cellular metabolic activity compared to mocktreated control cells (no ODN). The proliferation rate of the cells treated with the antisense ODN-PEG/KALA was significantly reduced relative to that of antisense ODN alone or the micelles containing mismatched ODN in the presence of serum. antisense ODN complexed with KALA (AS ODN/KALA) showed less extent of inhibition activity than the antisense micelle formulation (AS ODN-PEG/KALA), suggesting that PEG shell of the micelles not only enhanced the solubility of the polyelectrolyte complexes but efficiently stabilized the micelles in the presence of serum. Our previous studies also showed that the polyelectrolyte complexes between DNA and PEG-modified cationic polymers exhibited increased transfection efficiency compared to the complexes with unmodified polymer in the presence of serum (23, 24). This is probably because the flexible PEG chain can reduce the protein adsorption that induces interparticle aggregation. The same experiment set with mismatched ODN was also carried out but showed no significant effect on smooth muscle cell proliferation (data not shown). To study the effect of different core forming cationic polymers on the antiproliferation activity of the polyelectrolyte complex micelles, different cationic polymers including KALA, polyethylenimine (branched-PEI, MW25000), and protamine were used as counter polyions. As shown in Figure 8, all of the polycations demonstrated desired inhibition activities on the proliferation of smooth muscle cells. The polyelectrolyte complex micelles gener-

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Figure 8. Proliferation rate of smooth muscle cells treated with various polyelectrolyte complex micelles prepared by antisense c-myb ODN/PEG conjugate with different polycations, KALA, polyethylenimine (PEI), and protamine. The size and surface zeta-potential values of the different micellar formulations were similar to those of the ODN-PEG/KALA formulation. The experiment was performed under the same conditions as in Figure 7. The concentration of antisense ODN used in each formulation was 20 µg/mL. The experiment was carried out in triplicate.

ated by using protamine showed slightly lower inhibition activity, suggesting that either fusogenic activity of KALA or the endosomal disruption property of PEI could improve antisense activity of ODN. This result also demonstrated that any type of cationic polymers with or without functionality could be applied to the formulation of ODN-PEG/polycation complex micelle systems. In conclusion, this study demonstrates a novel formulation of antisense ODN for enhanced cellular uptake. ODN was conjugated to PEG to form a diblock copolymerlike structure via an acid-cleavable linkage, phosphoramidate. By combining the ODN-PEG conjugate with a fusogenic cationic peptide in aqueous solution, stable ODN-PEG/KALA micelles were produced. They were more readily transported within cells than ODN itself, which occurred probably by an endocytosis process. A therapeutic ODN, c-myb, exhibited far greater antiproliferation effect on smooth muscle cells when formulated the current micelle formulation. It can be envisioned that this novel formulation has a wide range of applications in intracellular delivery of ODN that has been the most challenging barrier for ODN therapeutics. ACKNOWLEDGMENT

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