Article pubs.acs.org/Biomac
Polymer Micelle with pH-Triggered Hydrophobic−Hydrophilic Transition and De-Cross-Linking Process in the Core and Its Application for Targeted Anticancer Drug Delivery Jianquan Fan,† Fang Zeng,*,† Shuizhu Wu,*,† and Xiaodan Wang‡ †
College of Materials Science and Engineering, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, People's Republic of China ‡ School of Pharmaceutical Chemistry and Chemical Engineering, Guangdong Pharmaceutical University, Guangzhou 510006, China S Supporting Information *
ABSTRACT: In this study, an novel amphiphilic block copolymer P[PEGMA-b-(DEMA-co-APMA)]-FA and its cross-linker uracil(CH2)6-uracil (U-(CH2)6-U) were synthesized and used as the targeted and pH-responsive nanocarriers for anticancer drug delivery. The hydrophobic block of the copolymer contains adenine (A) and tertiary amine moieties and the hydrophilic block is terminated with a targeting ligand folic acid (FA). Under neutral pH, the hydrophobic chain segments of the copolymer are cross-linked by U-(CH2)6-U through the A-U nucleobase pairing based on complementary multiple hydrogen bonding, and the copolymer forms stable micelles with their mean diameter of around 170 nm in water. While under acidic pH, the micelles dissociate as a result of protonation of tertiary amines and disruption of the A-U nucleobase pairing. Flow cytometry and fluorescent microscope observation show that, when loaded with an anticancer drug DOX, the micelles can preferably enter folate receptor (FR)-positive cancer cells and kill the cells via intracellular release of the anticancer drug. Cytotoxicity tests (MTT tests) indicate that the micelles with FA on their surfaces exhibit higher cytotoxicity toward FR-positive cells than those without FA. This study provides useful insights on designing and improving the applicability of copolymer micelles for other targeted drug delivery systems.
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INTRODUCTION In the last two decades, polymeric micelle formed by block copolymers has appeared as a new type of drug carrier,1−10 which is a macromolecular assembly composed of a hydrophobic core and a hydrophilic shell.11−26 Drug molecules can be incorporated into the micelle core through chemical conjugation or physical entrapment. Physical entrapment using hydrophobic interactions can be applied to many drugs, as most drug molecules possess a hydrophobic moiety(ies).13,18,22 Polymer micelles as drug carrier usually have several advantages. First, polymer micelles are formed typically in 50−200 nm with narrow size distribution, which is considered preferable for the stable, long-term circulation of the carrier system in the bloodstream because larger particles are actively captured in the reticuloendothelial system, while smaller particles are rapidly excreted from the kidneys. Second, they can improve the solubility and bioavailability of hydrophobic drugs in water. Third, they can have low toxicity through the employment of biocompatible polymer segments.13,18 Recently, stimuli-responsive micelles have attracted extensive attention for intelligent drug delivery in which the release of drugs can be regulated via an appropriate stimulus such as pH or temperature.26−39 Acidic pH as an internal stimulus is particularly appealing because tumor sites and the intracellular © XXXX American Chemical Society
compartments such as endosomes and lysosomes have a more acidic environment.23,32−39 The acidic pH has been considered as an ideal trigger for the selective release of anticancer drugs in tumors to achieve targeted drug delivery.34−39 Thus, nanoparticles or micelles based on acid-labile covalent bonds such as acetal and hydrazone are employed for drug delivery.27−43 For examples, Frechet and co-workers have exploited acetals as acid-labile linkages for pH-responsive polymer micelles.27−30 Kataoka et al.32 developed pH-sensitive polymeric micelles by attaching DOX to a polyaspartate block copolymer via an acidlabile hydrazone bond. Heller et al.36 have exploited orthoester as acid-labile linkages for pH-responsive micelles for drug delivery. Zhong et al.37 prepared block copolymer micelles with acetal moieties, which ensures acid-sensitive release of anticancer drugs. Our group also incorporated DOXs to nanoparticles via hydrazine linkages and obtained several prodrug systems that can release DOX under a lower pH environment such as in endosomes or lysosomes.43 On the other hand, polymer micelles are intrinsically unstable structures that can disassemble after infinite dilution that follows injection into the body.18,44 The stability of Received: September 11, 2012 Revised: November 9, 2012
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polymer micelles, which affects the stability of encapsulation of drug molecules, is a crucial condition for controlled drug delivery. Therefore, various approaches have been proposed to stabilize polymer micelles and thus encapsulation, and physical or chemical cross-linking has been used to stabilize polymer micelles.18,44−47 For cancer therapy, it is often more desirable to accomplish sustained drug release after micelles arriving at the pathological sites, which may enhance the therapeutic efficacy as well as reduce probability of drug resistance in cells.48 Compared to conventional chemical cross-linking, polymer micelles based on physical cross-linking have been found to be more sensitive to stimuli such as acidic pH, which offers an approach for designing drug delivery systems with rapid release at the tumor sites.49 In particular, physical crosslinking via hydrogen bonding is very sensitive to pH variation. Nucleobase pairing in biological systems is based on complementary multiple hydrogen bonding. In recent years, nucleobase pairing has been used for physically cross-linking or connecting polymer materials.50−61 For example, Long et al. synthesized side-chain-bound nucleobase polyacrylates and prepared novel polyacrylate adhesive with tunable cohesive strength through nucleobase pairing.50 Rowan et al designed thermally responsive polymers through nucleobase pairing induced assembly.56 Binder et al. synthesized polymers with terminal nucleobases and formed pseudoblock polymers through nucleobase pairing.60 Zhu et al. synthesized adenineterminated polycaprolactone and uracil-terminated poly(ethylene glycol) and formed block copolymer through nucleobase pairing for drug delivery.61 In the present study, we synthesized a novel block copolymer that can serve as a targeted and pH-sensitive anticancer drug delivery system. The schematic illustration for the delivery system and the molecular structure for the copolymer are shown in Schemes 1 and 2, respectively, and the synthetic routes for the cross-linker, the copolymer, and the control (with no FA) are displayed in Figures S1 and S2 in the Supporting Information. At physiological pH (∼pH 7.4), the copolymer behaves as an amphiphilic block copolymer; 85% of its hydrophobic block consists of hydrophobic segments of poly[2-(N,N-dimethylamino)ethyl methacrylate (DEMA)], and the other 15% contains segments with adenine (A) moieties that serve as the cross-linking sites and can form hydrogen bonds with the hydrophobic uracil(U)-containing cross-linker (U-(CH2)6-U) through the A-U nucleobase pairing. On the other hand, a PEG-containing segment, poly[poly (ethylene glycol) ethyl ether methacrylate] (PPEGMA), serves as the hydrophilic block, and a folic acid (FA) is covalently linked to one end of the hydrophilic block for targeting folate receptor (FR) positive cancer cells. Folic acid (FA), one of the most popular targeting ligands, retains high affinity for its receptor, even when linked to a variety of molecules; folate receptor (FR) is overexpressed in several human cancers, hence, FA can act as the targeting ligand for FR-positive cancer cells.62 Adding U-(CH2)6-U into the polymer solution causes the formation of water-dispersible micelles with cross-linked hydrophobic cores, which can encapsulate the anticancer drug DOX. At lower pH, the hydrophobic blocks of the copolymer undergo a transition from the hydrophobic to the hydrophilic state as a result of protonation of the tertiary amine moieties in side chains on the DEMAs segments, and this hydrophobic−hydrophilic transition in the micelle core initiates fragmentation of the micelle and leads to an increase in water uptake (bulk
Scheme 1. Schematic Illustration for the Micelle-Based Targeted Anticancer Drug with Nucleobase-Pairing CrossLinking in the Core
dissolution); in the meantime, the breakage of hydrogen bonds also occurs for the nucleobase pairs. The two transitions triggered by acidic pH result in the dissociation of the micelle and subsequent release of anticancer drugs, as shown in Scheme 1. There are several beneficial features of this micelle-based drug delivery platform with dual-transition capability. First, under physiological pH (∼7.4), the hydrophobic block (with unprotonized tertiary amines on the side chains) makes the block polymer readily form micelles, and the nucleobasepairing-based cross-linking ensures their stability, while under acidic pH, the dual-transition (protonization of tertiary amines and de-cross-linking in the micelle cores) triggers disintegration of the micelles and the subsequent release of anticancer drug. Second, under neutral pH, the optimized lengths of the hydrophobic and hydrophilic blocks in the polymer chain ensure drug encapsulating capacity and good aqueous dispersibility. Third, the PEG-containing hydrophilic block of the polymer ensures very low nonspecific protein adsorption, and incorporating targeting ligand (folic acid) onto the hydrophilic block affords the micelle active targeting capability.
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EXPERIMENTAL SECTION
Materials. Doxorubicin hydrochloride was purchased from Wako Chemicals (Japan). tert-Butyl carbazate, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC), poly(ethylene glycol) ethyl ether methacrylate (average Mn 475; PEGMA), and sulfo-N-hydroxysuccinimide (sulfo-NHS) were purchased from Sigma-Aldrich. 2-(N,N-Dimethylamino)ethyl methacrylate (DEMA), 3-bromo-1-propanol, 1,6-hexandiol, adenine, uracil, 4,4azobis(4-cyanopentanoic acid), benzyl chloride, Boc-L-aspartic acid 4benzyl ester, p-toluenesulfonyl chloride, and 4-dimethylamino pyridine (DMAP) were purchased from Alfa Aesar. The culture medium RPMI1640 and the FA-free culture medium RPMI1640 were obtained from Invitrogen. Triethylamine (TEA), hexane, heptane, ethyl acetate B
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Scheme 2. Structures of Copolymer P[PEGMA-b-(DEMA-co-APMA)]-FA and Schematic Illustration for Cross-Linking of the Copolymers by U-(CH2)6-U through Hydrogen Bonding between Uracil and Adenine Bases
(EtOAc), dichloromethane (DCM), methanol (MeOH), N,Ndimethylformamide (DMF), and dimethyl sulfoxide (DMSO) were distilled before use. All other chemicals used were of analytical reagent grade. Characterization. 1H NMR spectra of the samples were recorded on a Bruker Avance 400 MHz NMR spectrometer using CDCl3 or DMSO-d6 as the solvents at 25 °C. Absorbance measurements were carried out using a Hitachi U-3010 UV−vis spectrophotometer. The calibration curve of absorbance at 485 nm as a function of a series of DOX concentrations was produced using the same UV−vis spectrophotometer. The particle size was determined by DLS using a Malvern Nano-ZS90 particle size analyzer. Transmission electron microscopy (TEM) image was obtained using a JEM-100CXII transmission electron microscopy (Japan). Preparation of P[PEGMA-b-(DEMA-co-APMA)] and P[PEGMA-b-(DEMA-co- APMA)]-FA Micelles without Cross-Linking in the Core. First, the block copolymers P[PEGMA-b-(DEMAco-APMA)] and P[PEGMA-b-(DEMA-co-APMA)]-FA and the crosslinker U-(CH2)6-U were synthesized with the detailed procedures given in Supporting Information. The micelles of the two polymers were prepared using the membrane dialysis method. Briefly, the
synthesized P[PEGMA-b-(DEMA-co-APMA)] (20 mg) or P[PEGMAb-(DEMA-co-APMA)]-FA (20 mg) was dissolved in methanol (1 mL) at room temperature. After that, the polymer solution was added dropwise into 10 mL of deionized water under stirring with a magnetic bar. Then the methanol was removed by vacuum distillation with a rotary evaporator. The micelle solution was dialyzed against deionized water for 24 h (molecular weight cutoff, 1 kDa), during which the water was renewed every 4 h. Preparation of P[PEGMA-b-(DEMA-co-APMA)] and P[PEGMA)-b-(DEMA-co- APMA]-FA Micelles with A-U Nucleobase Pairing Cross-Linking in the Core. P[PEGMA-b-(DEMA-coAPMA)] or P[PEGMA-b-(DEMA-co-APMA)]-FA (20 mg) and the cross-linker (U-(CH2)6-U, 1.0 mg) were dissolved in methanol (1 mL) at room temperature. Then the polymer solution was added dropwise into 10 mL of deionized water under stirring with a magnetic bar. Afterward, the methanol was removed by vacuum distillation with a rotary evaporator. The micelle solution was dialyzed against deionized water for 24 h (molecular weight cutoff, 1 kDa), during which the water was renewed every 4 h. Drug Loading into Micelles with Cross-Linked Cores. Briefly, DOX·HCl (2 mg, 0.0035 mmol) and an equal molar amount of C
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Figure 1. Size distribution of the P[PEGMA53-b-(DEMA51-co-APMA9)]-FA micelles (with their cores cross-linked) determined by (A) DLS and (B) the TEM image of the micelles (negative staining with phosphotungstic acid was employed to outline the micelles). percentages of released DOX Er were determined according to the following equation:
triethylamine (TEA) were dissolved in DMF and added to a methanol solution of P[PEGMA-b-(DEMA-co-APMA)] or P[PEGMA-b(DEMA-co-APMA)]-FA (20 mg) and the cross-linker (U-(CH2)6-U, 1.0 mg). Then the mixture was added slowly to 10 mL of phosphatebuffered saline (PBS, pH 7.4). After being stirred for an additional 8 h, the solution was dialyzed against deionized water for 24 h (Molecular weight cutoff, 1 kDa), during which the water was renewed every 4 h. For determination of drug-loading content, the DOX-loaded micelle solution was lyophilized and then dissolved in DMSO. The UV absorbance at 485 nm was measured to determine the DOX concentration. Drug loading content and drug loading efficiency were calculated according to the following formula:
n−1
Er(%) =
Cn × V0 + ∑i = 1 (Ci × VS) Q
× 100%
where Cn represents the concentration of DOX in the nth sample; V0 is the whole volume of the release media (100 mL); VS is the volume of sampling aliquot (2 mL); and Q is the theoretical total DOX amount calculated based on the drug loading capacity. Cellular Uptake and Cytotoxicity. Three cell lines, HeLa (human cervical cancer cell, FA overexpressing), L929 (murine aneuploid fibro-sarcoma cell, normal cell, without FR overexpressing), and A549 (carcinomic human alveolar basal epithelial, without FR overexpressing) were incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO2. The cellular uptake experiments were performed using flow cytometry and fluorescent microscope. For the flow cytometry, HeLa, L929, and A549 cells (1 × 106) were seeded in six-well culture plates, respectively, and grown overnight. The cells were then treated with free DOX or P[PEGMA-b-(DEMA-co-APMA)] or P[PEGMA-b(DEMA-co-APMA)]-FA DOX-loaded micelles with (A-U) nucleobase pairing cross-linking in the core for 120 min. Thereafter, the cells were lifted and washed, and the DOX uptake was analyzed using a FACSCalibur flow cytometer (Beckman Coulter EPICS XL). A minimum of 1 × 104 cells were analyzed from each sample. For fluorescent microscope studies, HeLa cells (5 × 10 4) were seeded in 3.5 cm culture dishes and incubated overnight. The cells were treated with DOX-loaded micelles with (A-U) nucleobase pairing cross-linking in the cores, P[PEGMA-b-(DEMA-co-APMA)]-FA (the targeted prodrug) or P[PEGMA-b-(DEMA-co-APMA)] (the nontargeted prodrug) for 2 h. Then, the cells were washed with PBS buffer and treated with a drop of 0.4% trypan blue solution to quench the fluorescence of the micelles adsorbed on the outer cell membrane. Then the cells were imaged on an Olympus IX71 inverted fluorescence microscope equipped with a DP72 color CCD (excited at 465 nm). The cytotoxicity of the DOX-loaded (A-U) cross-linked micelles or the cross-linked micelles without DOX loading against HeLa, L929, and A549 cells was assessed by MTT assay. The cells were cultured in FA-free RPMI 1640 medium supplemented with 10% FBS (penicillin/ streptomycin 100 U/mL). The cells were seeded in 96-well plates at the cell population of 5000 cells/well. After 24 h of incubation in 96well plates at 37 °C, cells were washed with prewarmed PBS buffer, then the PBS was replaced with fresh medium containing free DOX, DOX-loaded (A-U) cross-linked micelles (the concentration of DOX = 0, 2, 4, 6, 8, 10 μg/mL), or copolymers P[(DEMA-co-APMA)-bPEGMA]-FA (content: 0−300 μg/mL) incubated for 48 h. Thereafter, the wells were washed with PBS buffer and incubated for another 4 h with RPMI 1640 medium containing 0.5 mg/mL MTT. After discarding the culture medium, 150 μL of DMSO was added to dissolve the precipitates and the absorbance was read with a Thermo MK3 ELISA reader at 570 nm. As for the assays, three independent experiments were performed for each concentration, and for each
drug loading(wt%) = (weight of loaded drug/weight of polymer) × 100% drug loading efficiency(%) = (weight of loaded drug/weight of drug in feed) × 100% Drug Loading into Micelles with Un-Cross-Linked Cores. DOX·HCl (2 mg, 0.0035 mmol) and an equal molar amount of triethylamine (TEA) were dissolved in DMF and added to a methanol solution of P[PEGMA-b-(DEMA-co-APMA)]/P[PEGMA-b-(DEMAco- APMA)]-FA (20 mg). Then the mixture was added slowly to 10 mL of phosphate-buffered saline (PBS, pH 7.4). After being stirred for an additional 8 h, the solution was dialyzed against deionized water for 24 h (molecular weight cutoff, 1 kDa), during which the water was renewed every 4 h. pH-Responsive Property of Micelles. To investigate the pHresponsive ability of micelles, the copolymer micelles were treated with different buffer solutions (pH 3.0−10.0) and the particle sizes were followed by DLS measurements. Briefly, 5 mL of DOX-loaded micelles was transferred into the different pH buffer solutions, respectively, and kept in a horizontal laboratory shaker maintaining a constant temperature and stirring (100 rpm) for 20 min, followed by DLS measurements. Moreover, the copolymer micelles were treated with buffers (pH 5.3 and 7.4), and the particle sizes were followed by DLS measurements at different time intervals. In Vitro Drug Release. In vitro DOX release assays were carried out according to the literature procedure,61 and the experiments were performed in triplicate at pH values of 7.4, 6.0, and 5.3 using 0.1 M PBS buffer (Na 2 HPO 4 −NaH 2 PO 4 ) and the acetate buffers, respectively. First, 5 mL of DOX-loaded micelles was placed in a dialysis bag with a molecular weight cutoff of 1000 Da. The dialysis bag was then immersed in 95 mL of the release medium (buffer) and kept in a horizontal laboratory shaker maintained at a constant temperature and stirring (100 rpm). At each predetermined time interval, 2 mL of the release medium outside the dialysis bag was taken out, and the same volume of each sample was replaced by the same volume of fresh medium. The DOX concentration in each withdrawn solution was determined using the UV-spectrophotometer at 485 nm against the predetermined calibration curve. The cumulative D
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Figure 2. 1H NMR chemical shift (400 MHz, in DMSO-d6) for NH (11.18 to 10.82 ppm) of the uracil moieties and chemical shift for NH2 (7.19 to 6.82 ppm) of adenine moieties in P[PEGMA53-b-(DEMA51-co-APMA9)]-FA and U-(CH2)6-U mixture (A-U = 1:1) as a function of temperature. Sample was allowed to equilibrate for 10 min at each temperature. independent experiment, the assays were performed in eight replicates. And the statistical mean and standard deviation were used to estimate the cell viability. Statistical Analysis. All data are reported as mean ± SD from three independent experiments, each performed in eight replicates; the statistical analyses were performed using GraphPad Prism 5.0 (t tests). A P-value
