Synthesis, Characterization, Antitumor Activity of Pluronic Mimicking

Doxorubicin (DOX), a topoisomerase inhibitor, is a widely used anticancer drug in ... Doxorubicin hydrochloride was purchased from Wako Chemicals (Jap...
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Bioconjugate Chem. 2008, 19, 525–531

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Synthesis, Characterization, Antitumor Activity of Pluronic Mimicking Copolymer Micelles Conjugated with Doxorubicin via Acid-Cleavable Linkage Yuhan Lee, Sung Young Park, Hyejung Mok, and Tae Gwan Park* Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea. Received October 15, 2007; Revised Manuscript Received November 12, 2007

Pluronic mimicking poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer having multiple hydroxyl groups in the PPO middle segment (core-functionalized Pluronic: CF-PLU) was synthesized for conjugation of doxorubicin (DOX). DOX was conjugated on the multiple hydroxyl groups of CF-PLU via an acid-labile hydrazone linkage (CF-PLU-DOX). In aqueous solution, CF-PLU-DOX copolymers self-assembled to form a core/shell-type micelle structure consisting of a hydrophobic DOX-conjugated PPO core and a hydrophilic PEO shell layer. The conjugated DOX from CF-PLU-DOX micelles was released out more rapidly at pH 5 than pH 7.4, indicating that the hydrazone linkage was cleaved under acidic condition. CF-PLU-DOX micelles exhibited greatly enhanced cytotoxicity for MCF-7 human breast cancer cells compared to naked DOX, while CF-PLU copolymer itself showed extremely low cytotoxicity. Flow cytometry analysis revealed that the extent of cellular uptake for CF-PLU-DOX micelles was greater than free DOX. Confocal image analysis also showed that CF-PLU-DOX micelles had a quite different intracellular distribution profile from free DOX. CF-PLU-DOX micelles were mainly distributed in the cytoplasm, endosomal/lysosomal vesicles, and nucleus, while free DOX was localized mainly within the nucleus, suggesting that CF-PLU-DOX micellar formulation might be advantageously used for overcoming the multidrug resistance (MDR) effect, which gradually develops in many tumor cells during repeated drug administration.

INTRODUCTION Doxorubicin (DOX), a topoisomerase inhibitor, is a widely used anticancer drug in the treatment of many types of cancer (1–4). However, systemic administration of DOX itself elicits severe cardiac toxicity due to the lack of ability to target cancer cells, and also shows the multidrug resistance (MDR) effect (5–7). To improve pharmacokinetic/dynamic profiles, enhance passive tumor targeting, and alleviate the MDR effect, various nanoscale DOX formulations such as polymeric nanoparticles, polymer conjugates, liposomes, polymeric micelles, and dendrimer conjugates have been reported to maximize its therapeutic efficacy (8–22). Polymeric and lipid nanoparticles containing DOX, when they are injected systemically, significantly alter their biodistribution profiles, thereby preferably targeting to the tumor tissue in a passive manner by the “enhanced permeation and retention (EPR)” effect (23). DOX has been physically encapsulated and/or chemically conjugated within poly(ethylene glycol) (PEG)-decorated biodegradable polymeric nanoparticles and micelles to prolong circulation time in the bloodstream as well as to increase the extent of extravascular accumulation in the tumor region. As an attempt to overcome the MDR effect that continuously pumps out intracellular free DOX molecules through the P-glycoprotein (P-gp) pump on the cell membrane, DOX was chemically conjugated onto water-soluble polymers, nanoparticles, and micelles via an acid-cleavable linkage, to change subcellular trafficking of DOX within cells (24, 25). Free DOX is taken up by cells via a passive diffusion mechanism, which is highly susceptible for the MDR effect. In contrast, DOX-conjugated nanoparticles and micelles are transported into cells via an * Corresponding author. T. G. Park, Tel +82-42-869-2621; Fax +8242-869-2610; E-mail address: [email protected].

endocytosis process, possibly reducing the concentration of cytosolic free DOX for the P-gp pumping action. It is conceivable that DOX molecules conjugated to nanoparticles and micelles are cleaved within the acidic endosomal vesicles and then transported into the cytosol, resulting in bypassing the MDR effect to some extent (26, 27). Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers (Pluronic) have been widely used as biocompatible drug carriers (28, 29). In aqueous solution, Pluronic copolymers self-assemble to form a spherical micelle above a critical micelle concentration and temperature. Pluronic micelles with ∼40 nm in diameter are composed of a hydrophobic PPO core and hydrophilic PEO corona. Various hydrophobic anticancer drugs have been physically entrapped within the PPO core of the Pluronic micelles to increase the drug solubility as well as enable passive targeting to the solid tumor. However, Pluronic copolymer micelles are not stable enough to maintain structural integrity for loading the moderately hydrophobic DOX within the inner core. This is partly due to the fact that hydrophobic selfassociation of PPO middle blocks in the core is not so strong in comparison to other polymeric micelles such as biodegradable poly(lactic acid)-b-PEG copolymer micelles. As a result, Pluronic copolymer micelles are easily disintegrated upon dilution below the critical micelle concentration after injection in the bloodstream, limiting their practical uses for systemic delivery of DOX. Nevertheless, it was recently demonstrated that Pluronic copolymer micelles, when administered together with various anticancer drugs, showed enhanced cytotoxic activities by sensitization of MDR cells (30–32). The sensitization effect to MDR cancer cells by Pluronic copolymers was attributed to the inhibition of P-gp activity by depletion of ATP. In this study, a Pluronic-mimicking copolymer having multiple hydroxyl groups on the PPO middle segment was

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synthesized and chemically conjugated with DOX via an acidlabile linkage. DOX was conjugated on the multiple hydroxyl groups on the PPO segment via a hydrazone linkage to form a more stable self-assembled micellar structure through π-π* interaction between polyaromatic ring structures in DOX buried in the core. It was expected that DOX was specifically released out in an intact form from the copolymer carrier under acidic physiological conditions such as an intracellular endosomal compartment within the tumor cells. The synthesized Pluronic F127 mimicking copolymer micelles were characterized using 1 H NMR, transmission electron microscopy (TEM), and light scattering. In vitro cytotoxicity and cellular uptake behavior of the DOX-conjugated copolymer micelles were evaluated for the MCF-7 human breast cancer cell line using free DOX as a control.

EXPERIMENTAL PROCEDURES Materials. Doxorubicin hydrochloride was purchased from Wako Chemicals (Japan). Epichlorohydrin, anhydrous 1,4dioxane, methoxy-poly(ethylene glycol) (mPEG) (MW: 5000), poly(propylene oxide) (PPO) (Mw: 2000), N,N′-dimethylethylenediamine, sodium chloride, triethylamine, anhydrous dimethylsulfoxide (DMSO), deutrium oxide (D2O), and dimethylsulfoxided6 (DMSO-d6) were purchased from Sigma-Aldrich (Milwaukee, WI). Sodium sulfate and sodium hydroxide were purchased from Junsei (Japan). p-Nitrophenyl chloroformate (p-NPC) was purchased from TCI (Japan). The MCF-7 cell line of human breast cancer cells was obtained from Korean Cell Line Bank (Seoul, Korea). Synthesis of PEO-PPO-PEO Triblock Copolymer Having Hydroxyl Groups on PPO Segment. Synthesis of EpoxideTerminated PPO and Epoxide-Terminated mPEG. Epichlorohydrin (7.44 g, 0.08 mol) was added dropwise to a mixture of PPO (40 g, 0.04 mol) and pulverized NaOH (5 g, 0.25 mol) in 50 mL of anhydrous 1,4-dioxane. The reaction mixture was heated to 65 °C and stirred magnetically for 6 h. The cooled reaction mixture was filtered, and the product was extracted in methylene chloride. The organic layer was dried over Na2SO4, concentrated by evaporation of methylene chloride, and further dried under vacuum. Synthesis of epoxide-terminated mPEG was also carried out similarly using the method described in the preparation of epoxide-terminated PPO. 1 H NMR (CDCl3): δ ) 3.88–3.08 (br, 2H of PPO), 3.08 (m, oxirane), 2.78 (m, OCH2), 2.42 (m, oxirane), and 1.16-1.03 (br, 3H of PPO). Synthesis of PPO Multiblock HaVing Multiple Hydroxyl Groups on the Chain. A PPO multiblock copolymer having multiple hydroxyl groups in the backbone was prepared by stepgrowth polymerization of epoxide-terminated PPO (21.1 g, 0.01 mol) and N,N′-dimethylethylenediamine (0.088 g, 0.01 mol) in refluxing ethanol (200 mL) for 40 h. After reacting for 40 h, an excess amount of N,N′-dimethylethylenediamine was added to introduce terminal amine groups in the PPO multiblock copolymer, and further reacted overnight in the refluxing solvent. After the reaction, the solvent was removed under vacuum and the remaining viscous solution was dissolved in methylene chloride (80 mL) followed by washing with saturated NaCl solution (10 mL). The organic phase was dried over MgSO4 and the solvent was evaporated under vacuum. The product was dissolved in deionized water, dialyzed to remove unreacted diamine, and lyophilized for 48 h. 1 H NMR (CDCl3): δ ) 3.88–3.06 (br, 2H of PPO), 2.3–2.25 (br, (CH3)CH2CH2N(CH3)), and 1.14-1.04 (br, 3H of PPO). Synthesis of PEO-PPO-PEO Triblock Copolymer. A triblock copolymer having multiple hydroxyl groups in the PPO backbone (core-functionalized Pluronic, CF-PLU) was prepared by reacting the amine-terminated multiblock PPO (15 g, 0.0036

Lee et al.

mol) to the epoxide-terminated mPEG (55 g, 0.01 mol) in refluxing ethanol (100 mL) for 40 h. The product was precipitated in excess cold diethyl ether. The triblock copolymer was purified by dialysis (molecular weight cutoff 6000–8000, Spectrum Laboratories, Inc., USA) in a cold room for 2 days, and lyophilized. 1 H NMR (D2O): δ ) 5.21–5.14 (br, 1H of lactide), 3.88–3.06 (br, 2H of PPO and PEO), 2.3–2.25 (br, (CH3)CH2CH2N(CH3)), and 1.2-1.05 (br, 3H of PPO). Synthesis of DOX-Conjugated PEO-PPO-PEO Copolymer via a Hydrazone Bond. Synthesis of Amine-ReactiVe PEO-PPO-PEO Copolymer. PEO-PPO-PEO copolymer was activated using p-NPC. Briefly, PEO-PPO-PEO copolymer (1 g, 0.071 mmol) was dissolved in distilled methylene chloride (20 mL), and triethylamine (0.049 mL, 0.071 mmol) was added in the solution. The copolymer solution was dropped into p-NPC (0.049 mg, 3 equiv) solution in methylene chloride (5 mL) for 1 h and reacted for 48 h with vigorous stirring. After evaporating methylene chloride, an excess amount of methanol was added to remove unreacted p-NPC. After removing methanol, the product was purified by extraction with sodium chloride saturated deionized water and precipitated in cold diethyl ether (-20 °C) and dried in vacuo. Conjugation of Hydrazine on PEO-PPO-PEO Copolymer. Amine-reactive PEO-PPO-PEO copolymer (1 g, 0.07 mmol) dissolved in methylene chloride (20 mL) was slowly dropped into hydrazine monohydrate (0.22 mL, 10 equiv) solution in methylene chloride (5 mL) and reacted for 24 h. After evaporating methylene chloride, the product was dissolved in deionized water and dialyzed against water and then lyophilized. Conjugation of DOX on PEO-PPO-PEO Copolymer. Hydrazine-conjugated PEO-PPO-PEO copolymer (100.58 mg) and DOX (10 mg, 1 equiv) were dissolved in anhydrous DMSO (10 mL). The reaction was kept for 48 h at 60 °C. Unreacted DOX and DMSO were removed using dialysis for 12 h at 4 °C against pH 9.0 distilled–deionized water adjusted with 0.1 M NaOH, and the product was lyophilized. All reactions were performed in a dark room. DOX conjugation was confirmed by gel permeation chromatography (GPC, Waters) detected by both an RI detector (RI-71, Shodex) and a UV detector (Waters 486 tunable absorbance detector, Waters) at absorbance 480 nm using chloroform as an eluant. The degree of substitution of DOX was determined using a spectrophotometer (Shimadzu UV-1601, Japan) at 480 nm. NMR Spectroscopy. Micelle formation of DOX-conjugated PEO-PPO-PEO copolymer via a hydrozone bond (core-functionalized Pluronic conjugated with DOX, CF-PLU-DOX) was confirmed by 1H NMR spectroscopy (Bruker Avance 400 spectrometer operating at 400 MHz). D2O and DMSO-d6 were used as solvents to compare the NMR spectra of DOX containing micelles and free DOX, which were prepared at a concentration of 0.1 (w/v) %. Transmission Electron Microscopy (TEM) Image Analysis. Transmission electron microscopy (TEM, CM-20, Philips, The Netherlands) was used to visualize the morphology of CF-PLU and CF-PLU-DOX micelles. For comparison, Pluronic F127 micelles were used as controls. Each sample was dropped onto a TEM copper grid covered with a carbon film (300 mesh, Ted Pella, U.S.A.) for 10 min and air-dried overnight. Size Distribution Using Dynamic Light Scattering. Dynamic laser light scattering (DLS) measurement was carried out using a Brookhaven laser light scattering instrument (ZetaPlus, Brookhaven Instruments Corporation, New York, USA), with a He-Ne laser operating at a wavelength of 677 nm at required temperature. A scattering angle of 90° was used for all measurements. The concentration of the sample was 0.005 (w/

Pluronic Mimicking Copolymer-Doxorubicin Micelles

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Scheme 1. Synthesis of Core-Functionalized PEO-PPO-PEO Copolymer (CF-PLU)

v) % in deionized water, and the solution temperature was equilibrated for more than 30 min before measurement. DOX Release Experiment. pH-dependent DOX release profiles from CF-PLU-DOX micelles were determined using a previously described method (27). Briefly, CF-PLU-DOX micelles (1 mg/mL) were prepared in pH 5.0 and pH 7.4 phosphate buffered saline (PBS) solutions. One milliliter of the solution was placed inside a dialysis membrane (molecular weight cutoff 3500, Spectrum Laboratories, Inc., USA) and dialyzed against 10 mL of pH 5.0 and pH 7.4 PBS solutions. The outer buffer solution containing released DOX was collected at predetermined time points and replaced with 10 mL of fresh buffer solutions. The amount of the released DOX was measured using UV–vis spectroscopy (Shimadzu, Japan) at a 480 nm wavelength and calculated on the basis of a calibration curve using DOX solutions with various concentrations as standards. The experiment was carried out in triplicate. In Vitro Cell Cytotoxicity, Confocal Image Analysis, and Flow Cytometry Studies. MCF-7 breast cancer cell line was used in this study. Cells were maintained in DMEM media containing 10% fetal bovine serum (FBS) in humidified air containing 5% CO2. Cells in each culture dish were exposed to various concentrations of free DOX and CF-PLU-DOX for 48 h and cell viability was examined. Cell viability was determined using the conventional MTT assay method. For confocal laser microscopy (Zeiss, Germany) images, cells on a slide glass were exposed to an equivalent DOX concentration of 5 µM for 15 min, washed out thoroughly, and visualized to observe the early stage of cellular uptake. The extent of cellular uptake was also quantified by flow cytometry (FACSCalibur, NJ) after exposure of the cells to the equivalent DOX concentration of 5 µM for 15 min.

RESULTS AND DISCUSSION Core-functionalized PEO-PPO-PEO (CF-PLU) was synthesized using an epoxide-amine reaction method as shown in Scheme 1. Two terminally functionalized epoxide groups in the precursor PPO chain were reacted with secondary amine groups of N,N′-dimethylethylenediamine as a chain extender, producing a multiblock PPO chain with six hydroxyl groups on the backbone. Using GPC, it was confirmed that two epoxideterminated PPO chains (Mw 2000) were chain-extended via the epoxide-amine reaction, producing the multiblock PPO chain having weight-average molecular weight of 4090 (number of

precursor PPO: 2). By conjugating an epoxide-terminated mPEG to the two terminal amine groups of the multiblock PPO chain, Pluronic-mimicking PEO-PPO-PEO copolymer with multiple hydroxyl groups on the PPO segment was synthesized. The weight-average molecular weight of the synthesized triblock copolymer was 14 348 (Mw of PPO segment 4090 and Mw of PEO segment 10 258) as determined by GPC. The balance of molecular weight between the PPO and PEO segments in the copolymer was similar to that in Pluronic F127. The copolymer had six hydroxyl groups in the PPO segment in total, resulting from four hydroxyl groups in the multiblock PPO chain (two epoxide-terminated PPO chains in series) and two hydroxyl groups in the two end-capped mPEG chains that had terminal epoxide groups before reaction. The multiple hydroxyl groups on the PPO segment of the CF-PLU copolymer were further modified with DOX via a hydrazone linkage, an acid-labile bond, using a sequential p-NPC-mediated hydroxyl-amine coupling reaction followed by an amine-ketone reaction as shown in Scheme 2. The degree of substitution (DS) for DOX was 67.9% (∼4.1 DOX per CF-PLU) as determined using a UV–vis spectrometric method. The conjugation of DOX to CFPLU was confirmed by GPC analysis as shown in Figure 1. CF-PLU-DOX as detected by UV absorbance at 480 nm was eluted earlier than CF-PLU that was detected by RI, suggesting that DOX was covalently conjugated onto the PPO backbone of CF-PLU. The formation of a core/shell-type micelle structure for CFPLU-DOX copolymer in aqueous solution was confirmed by 1 H NMR spectra using two different solvents (D2O and DMSOd6). For the spectra of CF-PLU-DOX copolymer using DMSOd6 as the solvent (Figure 2a), characteristic peaks of DOX (multiple peaks at 4.5–5.4 ppm and 0.8–1 ppm) were detected, indicating that the copolymer molecules were fully dissolved in the solvent while exposing the conjugated DOX in the solvent. In contrast, in the spectra taken in D2O as a solvent, the DOX peaks disappeared while characteristic PEO and PPO peaks still appeared, suggesting that DOX molecules conjugated to the hydrophobic PPO segment were buried within the core. The results supported that the CF-PLU-DOX copolymers selfassembled into core/shell-type micelles in aqueous solution. The effective diameter of CF-PLU-DOX micelles measured by dynamic light scattering was 164.9 ( 34.4 nm, while that of CF-PLU micelles was 88.5 ( 6.8 nm, indicating that the conjugated DOX in the core increased micelle size. The

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Scheme 2. Synthesis of Doxorubicin-Conjugated Core-Functionalized PEO-PPO-PEO Copolymer via a Hydrazone Bond (CF-PLU-DOX)

theoretical drug loading efficiency was 13.7% as calculated on the basis of the degree of substitution of CF-PLU-DOX (67.9%). To further investigate the morphology of core/shell-type CFPLU-DOX micelles, TEM images of CF-PLU-DOX or CF-PLU micelles were taken. Figure 3a,b clearly shows the core/shell structure of CF-PLU-DOX copolymer micelles in aqueous solution, which were composed of a dark core (average diameter: ca. 90 nm) surrounded by a relatively bright shell layer (average thickness: ca. 15 nm). At lower magnification (Figure 3c), tightly packed nanospheres having an average size of about 120 nm were observed. On the other hand, CF-PLU copolymer micelles were irregular in shape and showed an average size of about 90 nm, consistent with the size obtained from the DLS study. The irregular morphology with multiple nuclei might be caused by interfering hydrophobic interactions of PPO segments by multiple hydroxyl groups present in the synthesized PEO-PPOPEO copolymer. It was likely that water molecules associated with the hydroxyl groups could not be completely removed from

the core during the self-assembly process of the copolymers, resulting in the inner nuclei-like water pockets. Considering that Pluronic F127 micelles are about 40 nm in diameter in aqueous solution, the increased micelle sizes of CF-PLU and CF-PLUDOX could be attributed to the modified and core-functionalized PEO-PPO-PEO structure. It was likely that hydrophobic interaction and/or π-π* stacking of conjugated DOX molecules in the core were responsible for the self-assembling core/shell structure of CF-PLU-DOX micelles. This core/shell structure is promising for cancer therapy, because the PEG shell can provide a stealth property with prolonged circulation time in the bloodstream, while the stabilized inner core can prevent early DOX release from the micelles during the circulation. DOX release from CF-PLU-DOX micelles was evaluated at pH 5.0 and pH 7.4 conditions. CF-PLU-DOX micelles are expected to exhibit pH-responsive DOX release profile due to the acid-labile nature of the hydrazone linkage between DOX and PPO middle backbone. As shown in Figure 4, CF-PLU-

Figure 1. Gel permeation chromatography profiles before and after DOX conjugation. Solid line indicates CF-PLU copolymer before DOX conjugation using RI detector, and dotted line indicates CF-PLU-DOX copolymer after DOX conjugation using UV detector.

Figure 2. Micellization of DOX-conjugated PEO-PPO-PEO copolymer as confirmed by 1H NMR spectroscopy using (a) DMSO-d6 and (b) D2O as solvents. The size was determined by dynamic light scattering (DLS) analysis: (c) CF-PLU and (d) CF-PLU-DOX at a concentration of 0.05 (w/v) %.

Pluronic Mimicking Copolymer-Doxorubicin Micelles

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Figure 5. Cytotoxicities of CF-PLU-DOX micelles (O) and free DOX (•) using MCF-7 cell line.

Figure 3. TEM images of CF-PLU-DOX (a-c) and CF-PLU (d).

Figure 4. In vitro release profiles of DOX from CF-PLU-DOX micelles at pH 7.4 (O) and pH 5 (•).

DOX micelles showed much faster DOX release at pH 5.0 than at pH 7.4. At pH 5.0, CF-PLU-DOX micelles released out about 50% of the initially loaded DOX for the first 3 h, and liberated 84% after 26 h. However, at pH 7.4, the micelles released out only 15% of DOX for 3 h and less than 40% after 26 h. The accelerated DOX release in acidic pH condition is highly desirable for effective treatment of cancer. It is conceivable that CF-PLU-DOX micelles are passively targeted to the tumor tissue through the enhanced permeation and retention (EPR) effect with minimized DOX release during circulation in the bloodstream. There would be two beneficial effects for the acidsensitive DOX release: (1) after accumulation in the vicinity of the tumor cells, DOX could be cleaved and released from the CF-PLU-DOX micelles selectively in the acidic solid tumor microenvironment for passive cellular uptake (27, 29, 30); (2) and more importantly, intact CF-PLU-DOX micelles would be taken up by tumor cells through nonspecific endocytosis, and located preferentially in the acidic endosome compartments, where DOX could be cleaved, escaped from the endosome, and released out in the cytosol. The second cellular uptake mechanism of DOX conjugated to CF-PLU micelles via endocytosis

could bypass the MDR effect often observed for the cellular uptake of free DOX bypassive diffusion. Cytotoxicity effects of CF-PLU-DOX micelles were evaluated using the MCF-7 human breast cancer cell line. Cells were treated with free DOX or CF-PLU-DOX micelles with an equivalent concentration of DOX. As shown in Figure 5a, CFPLU-DOX micelles exhibited far greater cytotoxicity than free DOX. The inhibition concentration (IC50) of CF-PLU-DOX micelles was 1.2 µM, which is 15-fold lower than that of free DOX (18.5 µM). In addition, it was evident that the enhanced cytotoxicity of CF-PLU-DOX micelles was not due to the cytotoxicity of the carrier polymer (CF-PLU). The CF-PLU copolymer exhibited very low cytotoxicity (more than 90% of the cells survived) over the same polymer concentration range (data not shown). To further investigate the cellular uptake efficiency of CFPLU-DOX micelles, FACS analysis and confocal laser microscopy were used to quantitatively evaluate and visualize CFPLU-DOX micelles internalized within cells. As shown in Figure 6a, FACS analysis taken after treating for 15 min clearly showed that the cellular uptake extent of CF-PLU-DOX micelles was greater than that of free DOX, indicating that the enhanced cellular uptake of CF-PLU-DOX micelles was responsible for the increased cytotoxicity as shown in Figure 5a. Figure 6b,c shows confocal images of the cells treated with free DOX and CF-PLU-DOX micelles for 15 min. MCF-7 cells treated with free DOX showed that DOX was predominantly accumulated in the nuclei (left panel), while the cells treated with CF-PLUDOX micelles showed scattered intense small red dots and red fluorescence in the cytosol and nuclei (right panel). The different confocal images clearly suggested that the CF-PLU-DOX micelles were taken up by endocytosis-mediated cellular uptake, while free DOX was transported into cells via a passive diffusion mechanism. The intense DOX accumulation only in the nuclei for free DOX, not in the cytosol, was because intracellular DOX molecules in the cytosol were rapidly transported to the nucleus and avidly bound to the chromosomal DNA. In contrast, it appears that CF-PLU-DOX micelles were initially located within the endosome vesicles, releasing cleaved DOX in the cytosol region in a sustained manner. This suggests that the enhanced cytotoxicity for CF-PLU-DOX micelles was mainly due to their endocytic intracellular transport (33), increasing the extent of cellular uptake. Lastly, it should be noted that Pluronic-mimicking copolymers used in this study might also contribute to the sensitization of MCF7 cells, additionally influencing the extent of DOX-induced apoptotic cell death. Earlier studies reported that a physical

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ACKNOWLEDGMENT This study was supported by the grant from the National Research Laboratory project from the Ministry of Science and Technology, Republic of Korea.

LITERATURE CITED

Figure 6. Cellular uptake of CF-PLU-DOX micelles. (a) FACS analysis using MCF-7 cells treated with free DOX (green line) and CF-PLUDOX (red line). Confocal laser microscopy images for HeLa cells treated with (b) free DOX, and (c) CF-PLU-DOX micelles.

mixture formulation of DOX and Pluronic copolymers treated to MCF7 cells significantly changed its genome expression patterns: up-regulating the ATP-associated genes involved in the respiratory chain and genes involved in the apoptotic signal transduction pathway. In addition, Pluronic copolymers are known to suppress the overexpression of MDR-related genes for various non-MDR cancer cells (34, 35). Structurally similar to Pluronic copolymers, CF-PLU copolymers are also expected to exhibit the similar MDR suppression effect in MCF7 cells with initiation of apoptotic signal transduction. Thus, it is likely that the CF-PLU-DOX micelles might have significant effects on overcoming the MDR barrier in cancer cells. We are currently investigating the anticancer effect of CF-PLU-DOX micelles using MDR cells. In conclusion, a new class of Pluronic mimicking micelles was synthesized to release DOX in a pH-responsive manner. PEO-PPO-PEO copolymer having multiple hydroxyl groups on the PPO segment was synthesized and DOX molecules were conjugated via an acid-labile hydrazone linkage. The DOXconjugated copolymer self-assembled in aqueous solution to form tightly packed spherical micelles or micellar aggregates having a core–shell structure composed of a DOX core and PEG shell. The copolymer micelles released DOX to a greater extent under acidic condition. The cytotoxicity of CF-PLU-DOX micelles was 15-fold greater than that of free DOX for MCF-7 human breast cancer cells. The enhanced cytotoxicity was mainly due to the increased cellular uptake of DOX-conjugated micelles that were taken up by endocytosis. It was possible that core-functionalized Pluronic mimicking copolymers synthesized in this study could be similarly conjugated with other hydrophobic anticancer drugs such as paclitaxel. Self-assembled nanoscale CF-PLU micelles conjugated with potent antitumor agents are expected not only to improve solubility in aqueous solution, but also to enhance targeting capacity to the solid tumor.

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