Article pubs.acs.org/journal/abseba
Glutathione-Responsive Polymeric Micelles Formed by a Biodegradable Amphiphilic Triblock Copolymer for Anticancer Drug Delivery and Controlled Release Zhigang Xu,‡ Shiying Liu,‡ Yuejun Kang,* and Mingfeng Wang* School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore S Supporting Information *
ABSTRACT: This article presents a novel glutathione (GSH)-responsive poly(ethylene glycol)-b-polycarbonate-b-poly(ethylene glycol) triblock copolymer that self-assembles into micellar nanoparticles and simutaneously carry hydrophobic anticancer drugs such as doxirubicin. These drug-loading micelles show glutathione-triggered decomposition that leads to controlled drug release and cytotoxicity to cancer cells. This polymeric drug-carrier system integrates features of facile synthesis, stimuli-responsive drug release, good biocompatibility, and relatively low toxicity of the dissociated segments, thus representing a promising anticancer drug carrier for potential clinical applications. KEYWORDS: polymer, micelles, nanomedicine, cancer, controlled release
■
INTRODUCTION Polymeric micelles, usually formed by self-assembly of amphiphilic block copolymers in water, have been widely studied in nanomedicine for disease treatment including cancer therapy.1−10 The sizes of polymeric micelles can be facilely tuned from 10 to 100 nm by varying the lengths of hydrophobic and hydrophilic blocks of the copolymers, thus preventing quick renal clearance that is a major problem for small molecular drugs. In addition, these polymeric nanostructures tend to have higher stability and large drug-loading capacity (particularly for hydrophobic drugs) than liposomes. As a consequence, polymeric micelle such as Genexol-PM, a paclitaxel-loaded poly(ethylene glycol)-b-poly(lactic acid) micelle, has been approved for clinical use for cancer therapy.11 Doxorubicin (DOX)-loaded pluronic micelle has been in Phase III of clinical trial for gastric cancer.12 An ideal anticancer drug delivery system is expected to deliver the loaded drugs into tumor sites and then release the drugs there locally, thus reducing the potential side effect to normal tissues. Although most of the polymeric micelles developed so far are able to deliver the loaded drugs into tumor sites mainly via enhanced permeability and retention (EPR) effect,13−17 they lack controlled drug release properties. To that end, stimuli-responsive drug delivery systems have been developed by utilizing some intrinsic features of cancer cells, such as relatively low pH (∼5.0) and/or high intracellular concentration (∼10 mM) of glutathione (GSH).18−25 Recently, © XXXX American Chemical Society
drug-loaded polymeric micelles that are responsive to intracellular stimuli such as reductive species (largely GSH) and pH have been reported.26−35 Among the amphiphilic block copolymers used to form these stimuli-responsive drug delivery systems, polyethylene glycol (PEG) as the hydrophilic moiety is exclusively used. The role of PEG is not only to provide the solubility and colloidal stability in aqueous media but also reduce nonspecific uptake via the reticuloendothelial system, thus prolonging circulation time in body and enabling tumortargeting through the EPR effect.15,36,37 On the other hand, different hydrophobic polymers containing pH- and/or GSHresponsive moieties (e.g., hydrozone, disulfide) have been incorporated into block copolymers to impart stimuliresponsive properties.37−43 Herein, we report a new polymeric drug delivery system that combines GSH-triggered drug release with simultaneous selfdecomposition. The poly(ethylene glycol)-b-polycarbonate-bpoly(ethylene glycol) triblock copolymer, denoted as MPEGP(BHD-SS)-MPEG, consists of a hydrophobic disulfide-bond containing polycarbonate as the middle block, from which two hydrophilic PEG chains are tethered (Scheme 1). These welldefined amphiphilic polymers self-assemble into core−shell micellar structures (denoted as PCMs) in aqueous solution. Received: March 11, 2015 Accepted: May 21, 2015
A
DOI: 10.1021/acsbiomaterials.5b00119 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering Scheme 1. (A) Chemical Structure of MPEG-P(BHD-SS)MPEG and the Formation of PCM Micelles by SelfAssembly of This Block Copolymer; (B) Schematic Illustration of the GSH-Induced Drug Release Associated by Self-Decomposition of the Micellar Carriers
Scheme 2. Synthetic Route to MPEG-P(BHD-SS)-MPEG: (a) DMAP, in THF, Argon Atmosphere, 25 °C, 30 min; (b) MEPG, in THF, Argon Atmosphere, 25 °C, 12 ha
a
The number average degree of polymerization in the middle block P(BHD-SS) was determined based on 1H-NMR analysis.
Particularly, the polycarbonate block encapsulates anticancer drugs such as DOX through hydrophobic interaction. Furthermore, the polycarbonate block consisting of disulfide bonds enables GSH-induced release of the anticancer drugs inside tumor cells where GSH has significantly higher concentration than that in normal cells.28,44
■
Preparation of Disulfide Bond-Linked Polycarbonate Micelles (PCMs). Polycarbonate micelles (PCMs) were obtained by dialysis process. Typically, 10 mL of water was added into 1.0 mL of DMF solution containing 10 mg/mL of MPEG-P(BHD-SS)-MPEG under sonication, and then subjected to dialysis (MWCO = 3500 for the dialysis membrane) against DI water over 48 h to remove organic solvent, and then the volume of the PCMs micelles solution was adjusted to 20 mL with DI water, resulting in a micelle dispersion with a concentration of 0.5 mg/mL for further characterization. Measurement of Critical Micelle Concentration (CMC). CMC value of MPEG-P(BHD-SS)-MPEG block copolymer was determined using the following procedure: Pyrene (2.0 mg) was added in 10 mL of acetone first, and then 10 μL of pyrene solution was transferred into each cuvette. The acetone solvent was removed upon evaporation, and 3.0 mL of aqueous MPEG-P(BHD-SS)-MPEG solution with a series of concentrations in the range of 1.0−200 μg/mL was added into the pyrene-containing cuvette separately, resulting in a theoretically maximum pyrene concentration of 1.0 μM in water solution. The fluorescence spectrum was obtained using a 340 nm excitation wavelength, and the fluorescence intensity ratio of I371/I383 was used to obtain the CMC value. Loading DOX into the Polymeric Micelles. The DOX-loaded micelles were obtained by the following dialysis process. Typically, DOX·HCl (2 mg), triethylamine (20 μL), and MPEG-P(BHD-SS)MPEG (30 mg) were mixed into DMF (2.0 mL). The resulting mixture was stirred 2 h at 25 °C. After that, 20 mL of DI water was added to the mixture under sonication. The free DOX and organic solvent of DMF were removed using the same dialysis method described above. The concentration of resulting PCM-DOX solutions was adjusted to 0.5 mg/mL for further characterization. The amount of drugs in PCM-DOX micelles was determined by a Perkim Elmer LS-55 fluorescence spectrometer. The DOX-loading content (LC %) and the DOX entrapment efficiency (EE %) were obtained via the following two equations
EXPERIMENTAL SECTION
Materials. Polyethylene glycol monomethyl ether (MPEG, Mn ∼2000), bis(2-hydroxyethyl) disulfide (technical grade), glutathione (GSH), dimethylaminopyridine (DMAP), pyrene, DAPI, formalin solution, triphosgene, and anhydrous tetrahydrofuran (THF) were supplied by sigma-aldrich (USA). Doxorubicin (DOX) was obtained from Bei-Jing Hua-Feng Lian Bo United Technology (China). All the other solvents were supplied by Ctech Global Pte Ltd. (Singapore). The deionized water was supplied by Millipore (USA). Fluorescent dyes containing Alexa Fluor 633 (AF-633) phalloidin and Lyso TrackerRedDND-99, all cells culture reagents including fetal bovine serum (FBS), low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM), (1 × ) TrypLE Express Enzyme, penicillin/streptomycin antibiotics, and PrestoBlue cell viability reagent were provided by Life Technologies (Singapore). Synthesis of MPEG-P(BHD-SS)-MPEG. The triblock copolymer MPEG-containing disulfide bond of MPEG-P(BHD-SS)-MPEG were synthesized as follows (Scheme 2): Typically, bis(2-hydroxyethyl)
disulfide (BHD, 343 mg, 2.0 mmol), 4-dimethyl -aminopyridine (DMAP, 740 mg, 6.0 mmol), and triphosgene (BTC, 220 mg, 0.74 mmol) with a feed ratios (3:9:1) were completely dissolved in anhydrous tetrahydrofuran (THF) at 25 °C in a round-bottom flask under dry argon atmosphere, and the reaction mixtures were stirred for 0.5 h at 25 °C. Then MPEG (800 mg, 0.4 mmol) in THF were added into the reaction mixture and stirred continuously under dry argon atmosphere overnight. The resulting polymer was added into N,Ndimethylformamide (DMF) and precipitated into diethyl ether three times for purification. The obtained polymer was dried at 25 °C under vacuum over 48 h. A white powder of MPEG-P(BHD-SS)-MPEG block copolymer was obtained (0.25 g, Mn, GPC = 5.64 kDa, Mw/Mn = 1.34, Figure S3). The actual DP of BHD segment was determined to be 32 by 1H NMR results (Figure S1). B
LC% =
amount of drug in micelles 100% amount of micelles
EF% =
amount of drug in micelles 100% total amount of drug in feed DOI: 10.1021/acsbiomaterials.5b00119 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering In Vitro Release of DOX From the Polymeric Micelles. First, 2 mL of PCM-DOX solutions (1 × PBS, pH 7.4) was added into a dialysis bag (MWCO 3500) and soaked in a glass bottle containing 50 mL of 1 × PBS or [1 × PBS + 10 mM of GSH]. The capped glass bottle was immerged in a water bath at 37 ± 1 °C under gentle and continuous shaking. At default time points, 3 mL of the 1 × PBS solution was withdrawn from the glass bottle for characterization and then the same volume of fresh 1 × PBS buffer solution was added to the soaking media in the glass bottle to maintain the total volume as 50 mL. Then the concentration of released DOX drug at different time points was calculated according to the calibration curve of DOX (Figure S6) using fluorescence spectroscopy (the excitation wavelength is 488 nm and the emission wavelength is 586 nm). The total amount of released DOX was calculated using the formula reported previously25 and averaged from three repeating measurements. Stability of PCMs and PCM-DOX Micelles. The degradation process of PCMs was evaluated by the following experiment. Briefly, an aliquot (300 μL) of MPEG-P(BHD-SS)-MPEG (30 mg/mL in DMF) was diluted with 3 mL of deuterium oxide (D2O), to which GSH was added to reach a final concentration of 10 mM). After being stirred at 25 °C for 24 h, the mixture was centrifuged (5000 rpm, 5 min). The sediment was separated and redispersed in CDCl3 for 1H NMR characterization. To characterize the stability of the micelles using DLS, we dispersed the PCMs and PCM-DOX micelles into pH 7.4 PBS media containing different amounts of GSH. Then the micelle solution was incubated at 37 °C under shaking. The hydrodynamic size of the PCMs and PCMDOX micelles was acquired by DLS over different periods of incubation. Cytotoxicity by PrestBlue Assay. HeLa cells line was supplied by ATCC and cultured routinely in low glucose DMEM with 10% FBS and 1% penicillin/streptomycin antibiotics (37 °C, 5% CO2). We used PrestBlue assay to test the cell viability according to the manufacture’s protocols as previously discribed.25 First of all, the HeLa cells were seeded in the 96-well plate (the cell density is 1 × 104 cells/well) and were cultured overnight. Thereafter, the old medium was removed and new medium with blank PCMs, free DOX, and PCM-DOX at a varied concentration was added. After desired time, the medium was thrown away and then the cells were washed by PBS for three times. Thereafter, medium with PrestoBlue reagent was put into cells and incubated with 5% CO2 for 1 h at 37 °C. Simultaneously, medium with PrestoBlue reagents was also put into blank wells without cells as control. The absorbance at 570 and 600 nm were determined after 1 h by Plate Reader (Tecan Infinite M200). Each one has three parallel samples. The cell viability in the group without adding any drugs or micelles was set as 100%. Data in other groups were analyzed on the basis of the same protocol. Cellular Uptake. First, HeLa cells (the density is 1 × 104 cells/ well) were seeded in the 6-well plate and cultured overnight. Thereafter, free DOX and PCM-DOX solution were added into the above plate medium to make the concentration of DOX 50 μg/mL for 2 h. Cells were stained as previously described.25 Briefly, cells were fixed by formalin solution, followed by permeabilization with 0.1% Triton X-100 and blocked by 1% BSA. Then filamentous actin (Factin) was stained by AF-633 phalloidin while the nucleus was stained by DAPI. The 633, 488, and 405 nm lasers were applied to excite AF633 phalloidin, DOX, and DAPI, whereas emissions at 638−747, 490− 630 and 410−481 nm were collected, respectively (Objective: LD Plan-Neofluar 20x/0.4 KorrM27). Flow Cytometry. First, the HeLa cells were seeded in the 6-well plate with 1 × 104 cells/well and cultured overnight. Thereafter, free DOX and PCM-DOX were put into the medium to make the final concentration of DOX 10 μg/mL and cultured for 2 and 24 h, respectively. Finally, the cells were processed as previously reported.25 Briefly, cells were washed by PBS extensively and then detached by TrypLE Express. Detached cells were transferred, then centrifuged, finally resuspended in PBS and analyzed by flow cytometry (LSRII, BD Biosciences). FITC (excitation: 488 nm, emission: 500−560 nm) channel was selected to detect and around 10 000 gated cells were
tested. Blank cells without any drug or micelles were regarded as control. All data were processed by FlowJo software. Characterization. 1H NMR results were obtained at 300 MHz (Bruker, Germany) at 25 °C with the internal standard of tetramethylsilane. Transmission electron microscopy (TEM) results of samples were obtained using the JEM-1230EX (Japan), and the TEM samples were prepared by adding one drop of the solution sample on carbon-coated copper grids. The ζ-potentials and the size distribution of PCMs and PCM-DOX micelles were provided by dynamic light scattering (DLS, BI-200SM (USA)). The GPC results of polymers were obtained by gel permeation chromatography (GPC) (Agilent 1260, USA, styragel HT column), which was equipped with the waters 1260 pump and the agilent 1260 refractive index detector (RID). The solvent is THF and the eluent rate is 1 mL/min. The linear polystyrene was used as the standard in the calibration. Fourier transform infrared (FT-IR) results were obtained from PerkinElmer FT-IR spectrophotometer (USA). The Shimadzu UV-2450 spectrophotometer was employed to determine the UV−vis absorpition spectra (Shimadzu, Japan). Fluorescence spectra were provided by fluorescence spectrometer with the model of Perkim Elmer LS-55 (USA). The cellular images were obtained by a LSM 780 confocal laser scanning microscopy (CLSM, Carl Zeiss, Germany).
■
RESULTS AND DISCUSSION Synthesis of MPEG-P(BHD-SS)-MPEG Polymer. The synthetic route to MPEG-P(BHD-SS)-MPEG is depicted in Scheme 2. First, a polycarbonate homopolymer bearing disulfide moieties was synthesized via polycondensation of bis(2-hydroxyethyl) disulfide (BHD) and triphosgene (BTC) using 4-dimethylaminopyridine (DMAP) as the catalyst, followed by reaction with polyethylene glycol monomethyl ether MPEG) for end-capping acyl chloride in the polycarbonate over 12 h at room temperature. As shown in Figure S1, the 1H NMR spectrum clearly shows peaks characteristic of MPEG moieties (3.38 and 3.65 ppm) and units of BHD with signals at 4.4 and 2.96 ppm. The number-average degree of polymerization (DP) of BHD was calculated about 32 via calculating the integral ratio (peak 3 to peak 1, Figure S1). Further evidence with FT-IR (Figure S2) showed the strong peaks at 640 and 1731 cm−1, which correspond to the stretching of the −C−S− bonds and the carbonyl groups in MPEG-P(BHD-SS)-MPEG, respectively. The gel permeation chromatography (GPC) results of the obtained polymer showed a monomodal peak (Figure S3) corresponding to the molecular weight of 5.64 kDa (Mn) relative to linear polystyrene standards and a polydispersity of 1.34. Such relatively low polydispersity compared with other noncontrolled polymerization techniques, which was also observed by He and co-workers in PEG-polyurethane-PEG triblock copolymers,39 indicates the well-defined structure of the resulting block copolymer. Drug Loading and Stability Study of Polycarbonate Micelles (PCMs). Typically, self-assembly behavior of MPEGP(BHD-SS)-MPEG in water solution was obtained via fluorescence spectroscopy (pyrene as the probe). An apparent critical micelle concentration (CMC) of 33.3 μg/mL was obtained (Figure S4). Thus, micellar assemblies of this polycarbonate copolymer are expected to form at concentrations above the CMC. The corresponding polycarbonate micelles (PCMs) were prepared by a solvent exchange method.29 Furthermore, the anticancer drug of DOX was loaded, mainly depending on the hydrophobic−hydrophobic interaction between the DOX and P(BHD-SS) block of the polymer, into the PCMs to examine the DOX release from PCM-DOX. Compared with blank micelles, PCM-DOX shows C
DOI: 10.1021/acsbiomaterials.5b00119 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
show negligible change from 92.0 to 126.5 nm after being incubated in 1 × PBS with the period of 48 h, indicating the relatively high stability of PCMs under normal physiological conditions (Figure 2A). However, the average diameter of PCMs in a 1 × PBS solution with 10 mM GSH increased significantly from 92.0 to 235.5 nm over the same period. Similar phenomenon of size increase caused by GSH-triggered cleavage of the disulfide bonds and the consequent decomposition of the PCMs were also reported in other disulfide-bearing polymeric micelles.47−51 The mechanism behind this phenomenon is discussed further in the following section. Furthermore, we monitored the corresponding change of correlation functions of PCMs under different conditions by DLS (Figure 2B). The decay rate for the correlation function of PCMs micelles after incubation in 1 × PBS with 10 mM GSH for 48 h became very slower, indicating the formation of large aggregates.27 The formation of aggregates was presumably caused by the cleavage of disulfide bonds of the intermediate polymer and consequent shedding of the PEG shells, which is further discussed in the following section.43 However, the correlation function of PCMs under normal physiological conditions without GSH showed little change over the same period. Similar GSH-induced aggregation was observed in the PCM-DOX (Figure 2C, D). These results suggest that both PCMs and PCM-DOX have high stability under normal physiological conditions, but decomposition occurs at 10 mM of GSH, which triggers the dissociation of disulfide bonds. In Vitro Drug Release and Degradation Study. To demonstrate the feasibility of PCM-DOX micelles as anticancer drug delivery systems, we further obtained the in vitro release result using a simulated physiological condition (pH 7.4). Figure 3 shows the GSH-responsive release behavior of PCMDOX under simulated normal physiological condition (i.e., without GSH) in comparison to a reductive environment with 10 mM GSH. In the absence of GSH, the drug release within 40 h is minimal (
