Bioconjugate Chem. 2001, 12, 917−923
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Novel Polymer-DNA Hybrid Polymeric Micelles Composed of Hydrophobic Poly(D,L-lactic-co-glycolic Acid) and Hydrophilic Oligonucleotides Ji Hoon Jeong and Tae Gwan Park* Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, South Korea. Received April 17, 2001; Revised Manuscript Received June 27, 2001
Biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) was chemically conjugated to oligonucleotide (ODN) to form an amphiphatic structure which is similar to an A-B type block copolymer. A terminal end of PLGA was activated and reacted with primary amine-terminated ODN. The ODN/PLGA conjugates self-assembled in aqueous solution to form a micellar structure by serving PLGA segments as a hydrophobic core and ODN segments as a surrounding hydrophilic corona. Critical micelle concentration was determined by a spectroflurometric method. Atomic force microscopic observation revealed that the micelle size was around 80 nm. These micelles could release ODN in a sustained manner by controlled degradation of hydrophobic PLGA chains. Compared to unconjugated ODN, the ODN/PLGA micelles could be more efficiently transported within cells, presumably by endocytosis. This study proposes a potential delivery method of ODN into cells by forming hybrid ODN/PLGA micelles.
INTRODUCTION
Recently, much attention has been drawn to various amphiphatic block polymers, which can self-associate to form micelles in aqueous milieu. They can be utilized as potential drug carriers (1-6). Most polymeric micelles have a core-shell structure composed of hydrophobic segments as an internal core and hydrophilic segments as a surrounding corona (7). The size of polymeric micelles, approximately less than 100 nm in diameter, not only makes them ideal drug delivery carriers for escaping from renal exclusion and the reticuloendothelial system, but also provides them with enhanced endothelial cell permeability in the vicinity of solid tumors (8, 9). In particular, amphiphatic A-B type diblock copolymers demonstrated a fairly good thermodynamic stability in physiological solution by virtue of their relatively low critical micelle concentration (cmc) (10). Various polymeric micelles have been used to deliver anticancer drugs for passive targeting to solid tumors in vivo. For example, diblock copolymers composed of poly(aspartic acid) and poly(ethylene glycol) (PEG) have been used for delivering doxorubicin to solid tumors in a target specific manner by the “enhanced permenation and retention (EPR)” mechanism (11). More recently, a variety of biodegradable polymeric micelles, based on diblock copolymers composed of aliphatic polyesters and PEG, were synthesized. Poly(D,L-lactic-co-glycolic acid) (PLGA) has been most popularly used as a constituent of the diblock copolymers because it can be hydrolyzed in vivo into lactic and glycolic acid forms, which can be metabolized further into carbon dioxide and water (12). Oligonucleotides (ODNs) represent short DNA molecules, which have been tested for treating various genetic diseases. Oligomeric antisense DNA and RNA have been used to block the expression of specific proteins * Corresponding author. Tel: +82-42-869-2621, fax: +82-42869-2610, e-mail:
[email protected].
by hybridization with specific (target) mRNA sequences. The antisense effect of ODNs has been demonstrated in both in vitro cell culture (13, 14) and in vivo studies (1416), and their therapeutic potentials have been proposed (17). However, one of the most serious problems is to efficiently deliver them into cells (18, 19). To obtain the most potent antisense effect, the formulation of ODN with suitable carriers must be developed to facilitate cellular uptake. A variety of nonviral vectors such as cationic liposomes (20) and synthetic polymers (19) have been used for delivering antisense ODNs into mammalian cells. In this study, we conjugated c-myc antisense ODN as a model ODN to biodegradable PLGA to make an amphiphilic structure, which could form micelles in an aqueous phase, like A-B type diblock copolymers. The ODN/PLGA micelles were used as vectors for the delivery of antisense ODNs, which can facilitate the cellular uptake of ODN via endocytosis. Characteristics and cellular uptake behaviors of the ODN/PLGA polymeric micelles were investigated. MATERIALS AND METHODS
Materials. Poly(D,L-lactic-co-glycolic acid) (PLGA5010) having a lactic/glycolic molar ratio of 50/50 was obtained from Wako Chemicals (Japan). This polymer (nominal MW 10000) has a free hydroxyl and a carboxyl group at its terminal ends. Oligodeoxynucleotide (antisense c-myc ODN, CACGTTGAGGGGCAT), which has a primary amine group at the end of its 5′ phosphate, was synthesized, modified, and purified by Bioneer (Korea). Dicyclohexyl carbodiimide (DCC), N-hydroxysuccinimide (NHS), and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). All other chemicals were of analytical grade. Conjugation of Oligonucleotide to PLGA. PLGA (1.0 g, 0.1 mmol) dissolved in 5 mL of DMSO was activated by DCC (61.9 mg, 0.3 mmol) and NHS (34.5
10.1021/bc010052t CCC: $20.00 © 2001 American Chemical Society Published on Web 10/20/2001
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mg, 0.3 mmol) (PLGA/DCC/NHS stoichiometric molar ratio: 1:3:3). The activated polymer was precipitated in cold ethanol followed by drying under reduced pressure. ODN (1.0 mg, 0.2 µmol) dissolved in 100 µL of 5 mM sodium borate buffer (pH 8.0) was added to DMSO (0.9 mL). Insoluble salt particulates were removed from the buffer solution by centrifugation at 14000 rpm for 10 min. The supernatant solution was then dropped into the DMSO solution (1 mL) containing the activated PLGA (2 mg, 0.2 µmol) (stoichiometric ratio: 1:1). The reaction mixture was incubated at room temperature for 12 h and then dialyzed against deionized water for the removal of unreacted ODN and the generation of ODN/PLGA micelles. The concentration of ODN in the micelles was determined by measuring absorbance at 260 nm using a Shimadzu UV-Vis spectrophotometer (UV-1601, Japan). The formation of self-assembled micellar structure was characterized by gel permeation chromatography using Bio-Rad Bio-columns SEC 250 (300 × 7.8 mm) in an aqueous mobile phase with a flow rate of 1 mL/min. Elution profiles of micelles and ODN were detected by absorbance at 260 nm. Characterization of ODN/PLGA Micelles. Effective hydrodynamic diameter of the micelle was measured by dynamic light scattering at 25 °C using a dynamic light scattering photometer (Zeta Plus, Brookhaven Instrument Co., New York) equipped with He-Ne laser at a wavelength of 632 nm. The measurement was carried out in triplicate. Measurement of Critical Micelle Concentration (cmc). The critical micelle concentration was determined by a fluorescence probe technique using pyrene as a fluorescence probe, as described previously (21, 22). Pyrene dissolved in acetone was added to deionized water to make a concentration of 12 × 10-7 M, and acetone was subsequently removed by stirring for 5 h at room temperature. The final concentration of pyrene was adjusted at 6 × 10-7 M. The concentration of ODN/PLGA conjugate was varied from 0.05 to 500 mg/mL. Combined solution of pyrene solution and ODN/PLGA conjugate was equilibrated at room temperature in a dark room for 1 day before measurement. Fluorescence spectra were monitored at room temperature using a Shimadzu spectrofluorometer (RF-5301PC, Japan) at an excitation and emission wavelength of 339 and 390 nm, respectively. The spectra were accumulated with an integration of 2 s/nm. Atomic Force Microscopy (AFM). Five microliters of ODN/PLGA micellar solution (100 µg/mL) was dropped on a clean glass surface and air-dried at room temperature. The AFM analysis was carried out in a constant tapping mode by using Nanoscope III (Digital Instruments). A standard Nanoprobe silicon single-crystal lever was used as a cantilever and a constant force mode was used with a scan speed at 0.5 Hz. The scanned image was collected from a 2.5 × 2.5 µm area. In Vitro ODN Release. Five milliliters of the micelle solution containing 300 µg of ODN/PLGA conjugate in dry weight basis was put into a dialysis membrane (molecular cutoff: 50000). The dialysis bag was placed in a 50 mL conical tube filled with 4 mL of Tris-EDTA buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0), which was maintained at 37 °C with gentle shaking (100 rpm). The buffer was collected and replaced at desired interval times. The collected sample was filtered and stored at 4 °C until analyzed at 260 nm. Fluorescence Labeling of Micelles. A fluorescencelabeled oligonucleotide having a complementary sequence to antisense c-myc ODN (sense c-myc ODN) was used to
Jeong and Park
label the ODN incorporated in micelles by hybridization between sense and antisense ODNs. The sense ODN (ATGCCCCTCAACGTG) having a primary amine group at its 5′ end, was labeled by using rhodamine-NHS in PBS (pH 7.6) at room temperature for 12 h. Dialysis was carried out to remove unlabeled fluorescent dye. The labeled ODN was concentrated by using Centricon (Amicon). The labeled sense oligonucleotide was added to the micelle solution for labeling the ODN/PLGA micelles. The mixture was heated at 65 °C for 5 min and cooled in ice. Endocytosis of Micelles. NIH 3T3 murine fibroblast was purchased from Korea Cell Line Bank (Seoul, Korea) and were cultured in DMEM supplemented with 10% FBS, streptomycin at 100 µg/mL, penicillin at 100 IU/ mL, and 2 mM L-glutamine. Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere. Ten thousand cells per well were plated in a six-well plate (35 mm diameter, Nunc) containing collagen-coated coverglass in 1.5 mL DMEM with 10% FBS and incubated for 24 h before further experiments. After the replacement of the medium with fresh serum-free medium, the ODN/PLGA micelles hybridized with rhodamine-labeled sense ODN were added to attain a concentration of 80 µg/mL. After 3 h incubation, medium was discarded and the cells were washed four times with PBS. The cells were fixed by adding a fixing solution (0.2% glutaraldehyde, 0.5% formaldehyde in PBS) followed by incubation at 4 °C for 15 min. The cells were then washed once with PBS and visualized by a Zeiss LSM 510 confocal microscopy (Carl Zeiss, Germany). An argon/krypton mixed gas laser with excitation line at 568 nm was used to induce rhodamine fluorescence, which was observed by using 575-640 nm band-pass filter. Flow Cytometry. The efficiency of endocytosis was determined by using a flow cytometry. Ten thousand cells per well were plated and cultured in a six-well plate as described above. The medium was replaced with fresh serum-free medium prior to the addition of the micelles. The fluorescence-labeled ODN/PLGA micelles were then added to the cells to obtain a final concentration of 80 µg/mL. After 3 h incubation, the cells were washed four times with PBS, trypsinized, and fixed. The number of cells, which had endocytozed the micelles, was measured in a flow cytometry (FACSCalibur, Becton-Dickinson, Mountain View, CA). The cellular uptake efficiency of ODN/PLGA micelles was quantified by counting the number of cells within an arbitrarily selected gate region (50 < FL2-H < 350). RESULTS AND DISCUSSION
In our previous reports, various bioactive molecules and drugs, such as peptides, enzymes, and anticancer drugs, were directly conjugated to the terminal group of PLGA for achieving a sustained release from nanoparticles and microspheres (23, 24). Slow degradation of PLGA chain provided the controlled release of conjugated bioactive moieties over the desired period. More recently, doxorubicin was conjugated to the terminal end of PLGA in a block copolymer of PEG-b-PLGA to form polymeric micelles, where doxorubicin was chemically entrapped within a hydrophobic micelle core (6). It was found that chemically entrapped doxorubicin within PEG-b-PLGA could be transported within cells to a greater extent than doxorubicin alone, possibly due to an endocytosis process. In general, when hydrophilic synthetic polymers were linked to water insoluble PLGA in a diblock polymer structure, the A-B type diblock polymers self-assembled in aqueous solution to form micelles. We selected ODN
Polymer−DNA Hybrid Polymeric Micelles
Figure 1. The schematic diagram of micelle formation of amphiphilic conjugate of oligonucleotide and poly(D,L-lactic-coglycolic acid) in aqueous medium.
as a hydrophilic segment to conjugate to the terminal end of PLGA to form a novel hybrid polymeric micelle structure in aqueous solution (Figure 1). It is postulated that ODN, as the hydrophilic constituent part of ODN/ PLGA conjugate, can be more efficiently delivered within cells by an endocytosis mechanism, in contrast to ODN itself which is transported across the cell membrane by passive diffusion. An antisense ODN, c-myc, was used as a model ODN in this study. It is a single strand DNA with a molecular weight of 4633 and a nucleotide sequence of CACGTTGAGGGGCAT. The potential application of the antisense c-myc ODN for the suppression of smooth muscle cell proliferation was suggested in a previous study (25). Smooth muscle cell proliferation is believed to play a critical role in reoccurring the blockade of coronary artery after a balloon angioplasty treatment (restenosis). A reaction scheme for the conjugation of oligonucleotide and PLGA is shown in Figure 2. c-myc oligonucleotide terminally derivatized with a primary amine group was first dissolved in buffer solution. The solution was then added into DMSO containing preactivated PLGA with DCC and NHS. The volume ratio of aqueous to organic phase was adjusted to 1:10. The ODN/PLGA micelle was obtained and unreacted ODN was removed by dialysis against deionized water. The conjugation efficiency was determined by measuring the amount of ODN in ODN/ PLGA micelles via an UV spectrophotometric method and was about 75%. In addition, aqueous gel permeation chromatography was used to confirm the formation of ODN/PLGA micelles (Figure 3). The micelles were eluted in a void volume fraction at ca. 4.3 min, while ODN was eluted at ca. 9.3 min. This GPC result indirectly proves the formation of hybrid DNA/polymer micelles in aqueous solution. To determine the hydrodynamic volume of ODN/PLGA micelles, DLS was carried out as shown in Figure 4. It showed an effective diameter of 65.2 nm with a narrow size distribution. Atomic force microscopy was also used for the direct visualization of size and morphology of the ODN/PLGA micelles. In Figure 5, it can be seen that the micelles have a spherical shape with an average diameter of 80 nm. The morphological characters of ODN/PLGA micelles are quite similar to those of other reported synthetic polymeric micelles (26, 27). The diameter of micelles in the AFM image is slightly greater relative to that obtained from the DLS measurement. This was possibly caused by the change of micelle size during
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drying the spherically shaped hydrated micelles onto the flat surface for the AFM sample preparation. ODN/PLGA conjugate, consisting of a hydrophilic and negatively charged ODN segment and a hydrophobic PLGA segment, provided an opportunity to form selfassembled micellar structure in an aqueous phase. The conjugate has an amphiphatic structure like other synthetic diblock copolymers. The critical micelle concentration (cmc) of ODN/PLGA micelles was determined by using pyrene as a fluorescence probe, which partitioned into the hydrophobic PLGA core of micelles above the cmc value (21, 22). The cmc is an important physical property of the micelles and also indicates the stability of the micellar structure. Excitation and emission spectra of pyrene are shown at varying concentrations of ODN/ PLGA conjugate (Figure 6). In the excitation spectra, with increasing concentration of ODN/PLGA conjugate, greater fluorescence intensity was observed at around 339 nm (Figure 6A). It was reported that the excitation intensity of pyrene in an aqueous phase is very small at 339 nm, but it increases dramatically when pyrene is partitioned into a hydrophobic environment (10, 21). This suggests that the ODN/PLGA conjugate spontaneously formed micelles in aqueous solution that enabled pyrene to partition into the hydrophobic core of micelles. With increasing the concentration of the ODN/PLGA conjugate, a red shift, a characteristic feature of pyrene excitation spectra, was also noticed. The (0,0) band of pyrene shifted from 334 to 339 nm upon the partition of pyrene into the hydrophobic core of the micelles. Fluorescence intensity ratio at the two excitation wavelengths (I339/I334) as a function of ODN/PLGA conjugate concentration was used to determine the cmc value of ODN/ PLGA micelles according to the previous reports (10, 28). The cmc value determined by this method was about 5 mg/L as shown in Figure 7A. Figure 7B presents another method of determining the cmc value of micelles based on plotting the intensity ratio of I/III pyrene emission bands as a function of ODN/PLGA conjugate concentration. The cmc value of ODN/PLGA micelles was around 10 mg/L. Thus, an average cmc value of ODN/PLGA micelles calculated from the above two methods was determined as 7.5 mg/L. Although the cmc of ODN/PLGA micelles is higher than that of PEG-PLA diblock copolymer (cmc < 2 mg/L) (28), it is much lower than that of oligo(methyl methacrylate)-poly(acrylic acid) micelles (cmc ) 100 mg/L) (10). It is conceivable that negatively charged ODN chains in the shell layer surrounding the PLGA core repel each other by charge-to-charge repulsions, thus increasing the cmc value compared to those of synthetic polymeric micelles containing PEG as a shell layer. This result indicates that the ODN/PLGA micelles are relatively stable compared to conventional diblock polymeric micelles. ODN/PLGA micelles were subjected to incubation in aqueous solution to examine the release profile of ODN. Since the major constituent of the core region in ODN/ PLGA micelles is a biodegradable polymer, it was expected that ODN could be solubilized in an aqueous phase by slow degradation of PLGA chain. Random hydrolytic scission of the PLGA backbone would produce watersoluble oligo(lactic-co-glycolic acid) having a molecular weight of less than 1000. In our previous reports, it was shown that constant release of drugs could be attained from nanoparticles or microspheres prepared by PLGAdrug conjugates (23, 24). It can be seen in Figure 8 that ODN/PLGA micelles exhibited a near zero-order release over 50 days without any burst. The release profile suggests that ODN was released from the polymeric
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Figure 2. Conjugation scheme of amphiphilic conjugate of oligonucleotide and poly(D,L-lactic-co-glycolic acid).
Figure 3. Gel permeation chromatograms of ODN/PLGA micelle (a), and ODN (b).
Figure 5. AFM image of ODN/PLGA micelles on glass surface. The concentration of the micelle solution was 100 µg/mL.
Figure 4. Effective hydrodynamic volume of ODN/PLGA micelles analyzed by dynamic light scattering (DLS).
micelles by a degradation-controlled process of PLGA. Slow degradation of PLGA chains in the micelle core was likely to disintegrate the core structure of micelles, resulting in the release of ODN into the incubation medium. It should be noted that the released ODN fraction was not an intact ODN, but a mixture of ODNoligo(lactic-co-glycolic acid) conjugates which were soluble in water. The sustained release profile of ODN from ODN/PLGA micelles was not similar to that from PLGA microspheres physically encapsulating ODN molecules (19). The ODN/PLGA micelles exhibited a burst-free zero order release, whereas ODN containing PLGA microspheres showed a nonzero order release pattern with an initial burst release upon incubation. In the case of PLGA
microspheres, a depot formulation for subcutaneous and intramuscular tissues, it is difficult to precisely control the release rate of hydrophilic drugs such as ODN over a desired period, because diffusion and erosion processes are combined in a complicated manner to affect the release profile (24). In contrast, ODN/PLGA micelles that can be injected intravenously control the ODN release rate depending on the molecular weight and composition of PLGA to be conjugated. To visualize the endocytosis process of ODN/PLGA micelles into cells, a rhodamine-labeled sense ODN was hybridized with complementary antisense ODN present in the shell of micelles. This resulted in the micelle structure composed of double stranded ODN in the shell and PLGA in the core. These micelles were readily taken up by NIH3T3 mouse fibroblast cells as shown in Figure 9. It was found that the rhodamine labeled micelles were distributed over the entire intracellular space of the cytoplasm. In contrast, hybridized double stranded ODN labeled with rhodamine exhibited negligible fluorescence intensity within cells due to the limited passive diffusion of the ODN across the cell membrane. The confocal microscopic observation reveals that ODN can be
Polymer−DNA Hybrid Polymeric Micelles
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Figure 7. (A) The intensity ratio (339 nm/334 nm) of pyrene in the excitation spectra, (B) I/III band intensity ratio of pyrene as a function of logarithm of the ODN/PLGA conjugate concentration.
Figure 6. Excitation spectra of pyrene in the presence of ODN/ PLGA conjugate at a fixed emission wavelength of 390 nm (A), and fluorescence emission spectra of pyrene in the presence of ODN/PLGA conjugate at a fixed excitation wavelength of 339 nm (B). The concentration of pyrene was 6 × 10-7 M. The concentration of ODN/PLGA conjugate was varied from 0.05 to 500 mg/mL.
effectively transported within cells because cells recognize the self-assembled ODN/PLGA micelles as particulates. Thus, the increased cellular uptake of ODN/PLGA micelles was likely due to an endocytosis process. Presumably, a fluid phase pinocytosis played a pivotal role in the intracellular transport process of ODN/PLGA micelles, because the ODN surface of micelles was highly negatively charged. Due to charge-to-charge repulsion between micelles and cells, adsorptive endocytosis might have a minor role in the transport process. The fact that some micelles were located in the nucleus (dark and round area inside cells) suggested that the micelles can also be delivered into the nucleus. It was reported that PEO-PBLA[poly(β-benzyl L-aspartate)]-FITC micelles could be delivered into the nucleus using endothelial monolayer cells (27). Our results suggest the possibility of ODN fragments intracellularly released from the micelles to enter the nucleus and act as an antisense agent, interacting with specific sequence in mRNA of
Figure 8. In vitro release profile of ODN from ODN/PLGA micelles in Tris-EDTA buffer, pH 8.0 at 37 °C.
interest to block the expression of a target protein. The cellular uptake efficiency of the rhodamine-labeled ODN/ PLGA micelles was measured by a flow cytometry analysis. In an arbitrarily selected gate (50 < FL2-H < 350), the cellular uptake efficiency of ODN/PLGA micelles against fibroblast cells was 68.3% compared to ODN itself that showed a negligible cellular uptake of
