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Jun 3, 2015 - Vietnam National University, HoChiMinh City, Vietnam. •S Supporting ... ABSTRACT: In this report, a redox- and pH-sensitive poly(β-am...
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Redox- and pH-Sensitive Polymeric Micelles Based on Poly(β-amino ester)-Grafted Disulfide Methylene Oxide Poly(ethylene glycol) for Anticancer Drug Delivery Quang Nam Bui,† Yi Li,† Moon-Sun Jang,‡ Dai Phu Huynh,§ Jung Hee Lee,‡ and Doo Sung Lee*,† †

Theranostic Macromolecules Research Center, School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea ‡ Department of Radiology, Samsung Medical Center, Sungkyunkwan University School of Medicine and Center for Molecular and Cellular Imaging, Samsung Biomedical Research Institute, Seoul 135-710, Republic of Korea § Faculty of Materials Technology, National Key Lab. of Polymer and Composite Materials, HoChiMinh University of Technology, Vietnam National University, HoChiMinh City, Vietnam S Supporting Information *

ABSTRACT: In this report, a redox- and pH-sensitive poly(β-amino ester)-grafted disulfide methylene oxide poly(ethylene glycol) (PAE-gDSMPEG) was synthesized, and it showed not only a sharp pH-dependent assembly−disassembly transition but also a quick shell shading in a high concentration of reducing agent by Michael addition polymerization. 1H NMR, dynamic light scattering, and transition electron microscopy were combined to characterize the redox- and pH-responsiveness in various triggered conditions. The hydrophobic drug doxorubicin (DOX) was used as the model drug to investigate the encapsulation and delivery ability of polymeric micelles, in both in vitro and in vivo experiments. Notably, antitumor experiments in tumor-bearing mice showed that DOX-loaded polymeric micelles effectively enhanced the therapeutic efficacy in comparison to free-DOX. These results were further confirmed by histopathological examinations. Taken together, the results suggested that PAE-g-DSMPEG could be a potential hydrophobic drug delivery vehicle.

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INTRODUCTION In the battle against cancer, chemotherapy is one of the most common and conventional approaches, in which the strong hydrophobic drugs (e.g., doxorubicin, paclitaxel, and camptothecin) are used to help kill cancer cells. However, this therapeutic method suffers from nonspecific interactions, poor aqueous solubility, and multidrug resistance, which exist as major clinical barriers to their anticancer efficacy.1,2 A potential strategy to associate hydrophobic drugs with biocompatible delivery systems, like liposomes, polymeric nanoparticles, dendrimers, and micelles, has been widely developed to overcome these limitations and the serious side effects of chemotherapy. Among those drug delivery systems (DDS), stimulus-responsive biocompatible polymeric micelles have emerged as appealing carriers, with many superior features, including prolonged circulation time and increased accumulated drug concentration at the disease site by enhanced permeability and retention effect (EPR). These advantages have been indicated not only by many preclinical and clinical studies but also by several FDA-approved polymeric micelle DDS.3,4 Diverse physical (light, temperature, ultrasound, magnetic, mechanical, and electrical), chemical (solvent, ionic strength, electrochemical, and pH), and biological (enzymes and © XXXX American Chemical Society

receptors) conditions have been utilized for triggered-demicellization and the release of encapsulated cytotoxic drugs.7 Besides thermosensitive DDS, the pH-dependent polymeric micelle systems concurrently have been researched as ideal vehicles for selective delivery. Those micelle systems can be responsively destructed via reversible protonation−deprotonation core units or acid-labile bonds in the acidic environments. Because of the difference between the extracellular pH of normal tissues (pH 7.4) and of most solid tumors (lower than 7.2) or the pH changes in intracellular pathways (5.0−6.0 for endosomes and 4.0−5.0 for lysosomes), pH-sensitive polymeric micelles can enhance therapeutic activity of loaded drugs by spatially controlled release.8−14 For the last few decades, because of the overexpression of the reducing agent glutathione (GSH) in the intracellular matrix of tumor cells (concentration 100 to 1000 times higher than the extracellular environment), many groups have developed disulfide-containing polymeric micelle DDS in which the disulfide bonds can be broken in high GSH concentration environments to achieve fast intracellular Received: February 27, 2015 Revised: May 26, 2015

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Scheme 1. Mechanism of Drug Delivery and Release via the Destruction of Polymeric Micelles Triggered by pH and Reducing Agent

release.5,15−18 For more efficient delivery and precise release of the anticancer drugs, multistimuli-responsive polymeric micelle DDS that allow the spatial and temporal controlled release under a variety of triggered-environments have been developed.6 For example, Soppimath et al. synthesized pHand thermal-responsive copolymers based on poly(N-isopropylacrylamide), poly(N,N-dimethylacrylamide), and poly(10undecenoic acid), which can assemble stable core−shell nanostructures under physiological conditions to disassemble and release the encapsulated drugs in acidic environments.19,20 Additionally, Zhong et al. reported on redox- and pHresponsive micellar nanoparticles based on poly(ethylene glycol)-SS-poly(2,4,6-trimethoxybenzylidene-pentaerythritolcarbonate) (PEG-SS-PTMBPEC) copolymers for enhanced intracellular drug release under dual-stimuli environments.21 pH and magnetic cofunctionalization were also applied to prepare pH-responsive Fe3O4-cored nanoparticles that have efficient loading and pH-triggered releasing of anticancer drugs.22 Previously, our group prepared various series of biodegradable poly(β-amino ester) (PAE) by Michael-addition polymerization, which showed good pH-dependent micellization− demicellization transition because of the reversible protonation−deprotonation of tertiary amine groups on PAE polymer chains.9−13 Herein, we managed to prepare dual-sensitive polymeric micelles based on pH-sensitive core PAE and hydrophilic sheddable methoxy poly(ethylene glycol) (MPEG) shell. This amphiphilic (β-aminoester)-grafted-disulfide methoxy polyethylene glycol copolymer (PAE-g-

DSMPEG) can assemble into biocompatible micelles and be easily destructed under acidic conditions or under high concentrations of reducing agent, which results in the rapid release of therapeutic agents (Scheme1).

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MATERIALS AND EXPERIMENTAL SECTION

Materials. Methoxy poly(ethylene glycol) (MPEG, Mw = 5000 Da), succinic alhydride (SA), N,N′-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), cystaminedihydrochloride, Nhydroxysuccinimide (NHS), triethylamine (TEA), 4,4′-trimethylene dipiperidine (TDP), and 1,6-hexanediol diacrylate (HDD) were obtained from Sigma-Aldrich. Synthesis of Poly(β-amino ester)-Grafted Disulfide Methoxy Polyethylene Glycol. Poly(β-amino ester)-grafted disulfide methoxy polyethylene glycol (PAE-g-DSMPEG) was synthesized as shown in Scheme 2. First, MPEG-carboxyl (MPEG-COOH) was prepared via the reaction between MPEG (1 equiv) and SA (5 equiv), with DMAP (1 equiv) as the catalyst in anhydrous 1,4-dioxane for 24 h at room temperature (RT) under nitrogen protection. The solution was concentrated and precipitated in an excess of cold ether. Next, MPEGCOOH (1 equiv) was activated via NHS (10 equiv) and DMAP (0.05 equiv) in anhydrous N,N′-dimethylformamide (DMF) in the flask. DCC (10 equiv) in DMF was sequentially dropped into the flask. The reaction was kept for 24 h at RT and protected by nitrogen gas. The NHS-activated MPEG-COOH was collected by precipitation in cold ether. Modified MPEG further reacted with cystamine to form MPEGcystamine. In a flask with nitrogen protection, TEA was added to a solution of cystaminedihydrochloride in anhydrous dichloromethane (DCM) to remove hydrochloride (HCl), and the solution was vigorously stirred for 1 h before MPEG-NHS was added dropwise. After 24 h, the solution was concentrated and precipitated in cold B

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ether to obtain MPEG-cystamine. At every step, the products were confirmed by proton nuclear magnetic resonance (1H NMR). MPEG-cystamine (1 equiv of amine group), TDP (10 equiv), and HDD (11 equiv) were dissolved in a 1:1 volume ratio mixture of anhydrous dimethyl sulfoxide (DMSO) and chloroform (CHCl3), and the reaction was incubated at 55 °C for 3 days before precipitation in excess ether. The final product, PAE-g-DSMPEG, was characterized by 1 H NMR and gel-permeation chromatography (GPC), with CHCl3 as the eluent. Characterization of Polymers. The 1H NMR spectra were observed on Varian Unity Inova 500 MHz in deuterated chloroform (CDCl3) containing 0.03 v/v tetramethylsilane as a reference at RT. The molecular weight (Mn) and polydispersity index (PDI) were measured by GPC (model Fotecs NS2001, Shodex RI-101, K-804, K803, and K-802) using chloroform (CHCl3) as an eluent at 40 °C at a flow rate of 1.0 mL/min. The molecular weights were calibrated with poly(ethylene glycol) standards. Characterization of Micelles. The critical micelle concentration (CMC) was determined by using pyrene as the fluorescence probe, as previously described.10 An 84 μL aliquot of a stock solution of pyrene in THF (1 mg/mL) was added to 400 mL of PBS to get a final concentration of pyrene = 10−6 M. THF was eliminated by heating at 40 °C for 4 h. PAE-g-DSMPEG was dissolved and diluted in the pyrene solution to various concentrations. The pKa value of PAE-g-DSMPEG was calculated via a titration curve.12 Briefly, 50 mg of polymer was dissolved in 50 mL of deionized water at a pH lower than 4. By adding 50 μL of 0.1 N NaOH aqueous solution, the pH was measured to obtain the titration curve. The pKa

was calculated from the derivative values of the titration curves, which correlated with the inflection point. The micelle size was measured by dynamic light scattering (DLS) (Zetasizer, model Nano-ZS90) with a laser at 633 nm and a digital correlator. The scattering angle was fixed at 90°, and the temperature was 25 °C. PAE-g-DSMPEG polymeric micelles (PMs) were prepared in PBS solution (polymer concentration was 1 mg/mL) at a pH lower than 4. The micelle size was recorded at various pH values. The morphology of polymeric particles was obtained via transmission electron microscope (TEM) (JEOL USA, JEM-2100F). TEM sample was prepared by two-droplet method. A drop (10 μL) of micelle solution (concentration 1 mg/mL) was placed on a copper grid, and the grid was partly dried by a filter paper. Sequentially, the grid was stained by a small drop of 1% (w/w) tungstophosphoric acid solution, and excess stain solution was removed by filter paper. The stained grid was allowed to dry at RT before being scanned by TEM without any further treatment. Encapsulation of DOX in PAE-DSMPEG Micelle. DOX-loaded PMs were prepared by the film-hydration method.10 A stock solution of DOX in THF was prepared. DOX.HCl (1 mg) was dispersed in THF, after which TEA (2 equiv of HCl) was added, and the solution was stirred overnight in the dark to remove the HCl. After filtration to remove the TEA.HCl salt, 10 mg of polymer was dissolved in the above-mentioned THF solution. The organic solvent was removed by a rotary evaporator to form the DOX/polymer thin film. Subsequently, the DOX-loaded micelle was formed by adding 10 mL of pH 7.4 PBS with gentle shaking. The unloaded doxorubicin was removed using a 0.2 μm syringe filter, and the drug loading efficiency was analyzed by C

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Figure 1. 1H NMR spectra of PAE-g-DSMPEG. the absorption at 495 nm using UV−vis spectrometry (Optizen 322OUUV). Drug loading efficiency (DLE) and drug loading content (DLC) were calculated with following equations: DLE (%) =

weight of loaded drug × 100% weight of feeding drug

DLC (%) =

weight of loaded drug × 100% weight of micelle

thrice with PBS before being mounted on the slides. Cellular uptake was monitored by confocal laser-scanning microscopy LSM700 (CarlZeiss) X400. The scale bar represents 20 μm. Therapeutic Efficiency Experiment. The antitumor efficiency of the micelles was assessed in subcutaneous tumor-bearing mice. BALB/ c nude mice (6 weeks old, Oriental Bio, Seoul, Korea) were managed according to the guidelines of the American Association for the Accreditation of Laboratory Animal Care (AAALAC). The animal model was prepared by subcutaneous injection of hepatocellular carcinoma HepG2 cells (1 × 107 cells per mouse) into the mice. When the tumor volume reached 100−200 mm3, mice were randomly classified into three different experimental groups as follows: (i) normal saline (control group), (ii) free-DOX at 2 mg of DOX per kg of body weight and (iii) DOX-loaded PMs at 2 mg of DOX per kg of body weight. The tumor-bearing mice were intravenously injected with 200 μL of treatment solution through the tail vein once every 2 days for 14 days. The therapeutic efficiency was evaluated by recording the tumor volume and the body weight of each mouse for 20 days. The tumor length and width (the largest and smallest diameters respectively) were measured with a digital caliper, and the tumor volume was calculated according to the following formula: (width2 × length)/2. At 20 days postinjection, the mice were sacrificed, and the tumor tissues were histologically evaluated via hematoxylin and eosin (H&E) and TUNNEL staining (Promega Corp, WI).

In Vitro Release of DOX. A solution of DOX-loaded PMs was added to a cellulose dialysis membrane (MW cutoff = 3500 Da) and placed in PBS solutions of different pHs (7.4 and 6.4), with or without DTT (10 mM), in a shaking incubator (companion BS-21) at 37 °C. The PBS solution was changed at different time intervals. The drug concentration in the media was measured by UV−vis at 495 nm and used to calculate the accumulated release. The standard was set using different DOX concentrations from 0.01 to 500 μg/mL. Cytotoxicity Test. The cytotoxicity of polymeric micelles was evaluated in human hepatocellular carcinoma HepG2 cells. The cells were cultured in 96-well plates (density = 1 × 104 cells/well) in MEM (Gibco, Grand Island, NY) containing 10% (v/v) of FBS (Gibco, Grand Island, NY) and 1% (w/v) of penicillin−streptomycin overnight (37 °C, 5% CO2). Subsequently, DOX-loaded PMs samples or empty micelle samples were added into the wells to achieve different final concentrations. At 24 h post-treatment, Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) was used to assess cytotoxicity. Each sample was treated with 10 μL of CCK-8 solution at 37 °C for 4 h before the absorbance of CCK-8 at 450 nm wavelength was assayed with a microplate spectrophotometer (xMark, BIO-RAD). Cellular Uptake Assay. The cellular uptake of DOX-loaded PMs was estimated with HepG2 human hepatocellular carcinoma cells. In brief, HepG2 cells were seeded on coverslips within 6-well plates (1 × 104 cells/well) in MEM (Gibco, Grand Island, NY) containing 10% (v/v) of FBS (Gibco, Grand Island, NY) and 1% (w/v) of penicillin− streptomycin overnight (37 °C, 5% CO2). Afterward, the cells were treated with free DOX or DOX-loaded PMs (final concentration of DOX = 10 μg/mL) for various treatment times. Following this, cells were fixed with 3.7% formaldehyde in PBS for 10 min and washed with PBS. The cells were incubated with DAPI for 10 min and washed

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RESULTS AND DISCUSSION Synthesis and Characterization of PAE-g-DSMPEG. The hydroxyl functional group of MPEG was modified via SA, which was subsequently activated and coupled with cystamine by amide bond formation. The resulting modification of each step was examined by means of 1H NMR, which was reported in Figure S1. By comparing 1H NMR spectra of MPEGCOOH, NHS-activated MPEG, and MPEG-cystamine (Figure S1), the appearance of the peaks f and g (at 2.81 and 2.7, respectively) confirmed the successful synthesis of MPEGcystamine. PAE-g-DSMPEG was synthesized by Michael-type polymerization. The results of 1H NMR and GPC were used to confirm the chemical structure and molecular weight of the copolymer. D

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selected to calculate the graft ratio of MPEG. Integrated intensity ratio denoted that MPEG graft ratio was 11.8 mol %. Combining 1H NMR with GPC data, x and y value came out to be 7 and 1, respectively, indicating that PAE-g-DSMPEG contains one MPEG in its structure. Properties of Polymeric Micelles. The critical micelle concentration (CMC) was analyzed by fluorescence spectrometry with pyrene as probe. Pyrene is a highly hydrophobic molecule, which has a poor solubility in water (2−3 μM).27 With the presence of micelles, pyrene is primarily solubilized in the hydrophobic core of those micelles. By monitoring fluorescence intensity of probe with different environment around the probe from water to hydrophobic regions of micelles, the incorporation of pyrene in micelles can be reflected or CMC can be determined.27,28 Figure 2A reported the change in the intensity ratio of the first vibrational band to the third vibrational band (I338/I334) versus the polymer concentration. At the concentration below CMC, I338/ I334value remained virtually constant whereas above CMC, I338/I334increased substantially reflecting the formation of micelles. The CMC was determined as the concentration corresponding to the crossing point of the two tangents, which came out to be 0.4 mg/L. This small value of CMC also indicated that the micelle structure of PAE-g-DSMPEG may remain to be stable at low concentrations. Subsequently, pyrene was used for examining pH-dependent micellization−demicellization transition of PAE-g-DSMPEG. A polymer solution (concentration = 1 mg/mL in pyrenecontaining PBS solution) was adjusted to various pHs from 6.0 to 7.8. In Figure 2B, I338/I334was plotted as a function of pH which signified intensity of pyrene in divergent microenvironments. At acidic pH, the ionized tertiary amine groups induce micelle dissociation via both electrostatic repulsion and hydrophilic−hydrophobic transition of PAE backbone. As the result of that, pyrene in an aqueous environment shows a low value of fluorescence intensity ratio. Indeed, Figure 2B presents that I338/I334 ratio essentially remained at low level in the pH range from 6.0 to 6.6. However, the value of I338/I334 increased considerably from pH 6.8 and achieved two times higher compared with that of the low pH state. This fluorescenceincreasing phenomenon can be explained by the deprotonation of tertiary amine groups, which lead to micelle assembly or the incorporation of fluorescence probe in the hydrophobic core of PMs. This data suggests that the self-assembly behavior of PAEg-DSMPEG is to form unimers at acidic pH and micelles at physiologic pH and higher. The pH buffer capacity of PAE-g-DSMPEG was estimated by the titration method. The pH change of polymer in water solution (concentration = 1 mg/mL) against the addition of OH− was recorded in Figure 3. The plateau area of titration curve, in which the pH of solution increased slowly as more NaOH was added, indicates the buffer range of PAE-gDSMPEG. The pKa of PAE-g-DSMPEG was determined by the first derivative of curve formula to be 6.37. As shown in Figure 3, the PAE-g-DSMPEG buffer bar showed a slower increase in pH when it was higher than 6, which demonstrates the deprotonation of the tertiary amine groups. It also confirms that PAE-g-DSMPEG exhibits a sharp buffering capacity.9,12 The DLS data (Figure 4A) shows the changes in hydrodynamic size of micelles under different conditions. At a pH lower than 6.8, the polymer was soluble or no particle was detected because of the protonation of tertiary amine groups on PAE backbone. With increasing pH to greater than 6.8, the

Figure 2. (A) Plot of I338/I334 versus the logarithm of polymer concentration at pH 7.4 (B) versus that at various pH.

Figure 3. Titration curve of PAE-g-DSMPEG.

From GPC analysis (Figure S2), the average molecular weight and polydispersity index were determined to be 8700 Da and 1.12, respectively. In the 1H NMR spectrum (Figure 1), proton peak b of MPEG (4H, −CH2−CH2−O−) and proton peaks m and n of the TDP unit (6H, −CH2−CH2−CH2−) were E

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Figure 4. (A) Particle size of PAE-g-DSMPEG micelles at various pH values, (B) the particle size change of PAE-g-DSMPEG without (w/o) or with 10 mM DTT at pH 7.4, (C) TEM image of PAE-g-DSMPEG micelles, scale bar 100 nm, and (D) digital image of PAE-g-DSMPEG with and without 10 mM DTT at 7.4 after 60 min.

particle size and PDI of micelles at pH 7.4 were recorded as 60.2 ± 0.75 nm and 0.075, respectively. This micellization− demicellization behavior is in agreement with the alternation shown in Figure 2B, in which there was a remarkable increase of I338/I334 at pH higher 6.8. The morphology of polymeric particle was also observed via TEM (Figure 4C), and the size of the particle was shown to be about 40 nm, which is slightly smaller than that from the recorded data of DLS. This is likely due to the shrinkage of the polymeric particle during TEM preparation. The redox-responsive property of the particle was analyzed via the addition of the reducing agent, DTT (concentration = 10 mM), to the PMs solution. Then, the changes in particle size were recorded by the DLS instrument at various time intervals. A fast aggregation was monitored, in which the average particles size was increased from 60.2 to 138.6 nm just after 5 min and up to 196.5 nm after 60 min. In contrast, the samples without DTT treatment remained unchanged (Figure 4B). This phenomenon is the result of MPEG shell detachment caused by the cleavage of disulfide bonds, which causes clustering of the unprotected hydrophobic PAE core.16 Additionally, the turbidity change of micelle solution was captured and reported

Figure 5. In vitro drug release of drug-loaded PMs at pH 7.4 (black), pH 6.4 (blue), pH 7.4 with 10 mM DTT (red), and pH 6.4 with 10 mM DTT (green).

formation of PMs occurred as the deprotonation of amine groups made the backbone more hydrophobic. The average F

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Figure 6. In vitro cytotoxicity of empty PMs and DOX-loaded PMs at different concentrations in HepG2 cells after 24 h incubation.

Figure 8. In vivo therapeutic efficacy of DOX-loaded PMs (n = 10). (A) Tumor growth rate and (B) change in body weight of tumorbearing mice treated with saline (black), free DOX (red) and DOXloaded PMs (blue).

intermediate disulfide bonds, which may induce the micelle destabilization. Drug Loading and Triggered Release. Drug-loaded PMs was prepared by the film-hydration method. The drug loading content was designed to be 10 wt %. The calculated drugloading efficiency and drug-loading content were 49.6 and 4.96%, respectively. Drug-release behavior of DOX-loaded PMs was investigated and reported in Figure 5. At pH 6.4, the release rate was shown to be higher than that at pH 7.4 owing to the fast demicellization reported and discussed above (Figure 2A). This profile indicates that PAE-g-DSMPEG possesses pHtriggered release performance similar to our previous PAE micellar system.12,13 When DOX-loaded PMs was placed under high DTT concentration conditions, the cumulative curve was almost alike to that at pH 6.4 with about 6% lower. This result can be explained by the cleavage of disulfide bonds or the shedding of MPEG shells, which cause the DOX-PAE cores to become more hydrophobic and quickly aggregate and block the diffusion of DOX. Remarkably, PAE-g-DSMPEG released DOX rapidly in an acidic environment (pH 6.4) with the presence of reducing agent DTT, and up to 91.7% of DOX was released after 4 h. In comparison with single-trigger conditions (pH 6.4 or pH 7.4 + DTT), DOX-loaded PMs in pH 6.4 + DTT showed a two times higher cumulative percentage, which

Figure 7. CLSM images of HepG2 cells incubated with Free-DOX for (A) 1 h and (B) 12 h and with DOX-loaded PMs for (C) 1 h and (D) 12 h. Scale bar: 20 μm.

in Figure 4D, which obviously shows the contrast in two cases (with and without DTT addition). At pH 7.4 without reducing agent, the solution maintained high clarity (clear vial) after 60 min. However, the vial with DTT treatment quickly became strongly turbid, which reflects the assembly of hydrophobic PAE into large particles. Combined with size-change results in Figure 4D, hydrophilic shells of PAE-g-DSMPEG particles show the ability of being shedded via reduction at the G

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Figure 9. H&E staining and TUNEL assay of isolated tumor sections.

and DOX-loaded PMs, the drug-encapsulating polymeric nanoparticles showed a higher therapeutic efficacy, with smaller tumor volume increment. Nevertheless, free DOX was less effective because of the lack of protection from accumulation at organs or quick clearance from the body, which was provided by the polymeric vehicles.1,2 The increased therapeutic efficacy of DOX-loaded PMs proved the effective delivery of DOX to the tumor site, which was the result of the prolonged circulation time that was achieved because of the hydrophilic shell’s protection and demicellization in the acidic extracellular environment of the tumor or both the pH- and redox-triggered intracellular environment, as expected from the release profiles (Figure 5). Furthermore, Figure 8B shows that the treatment with DOX-loaded PMs had a better safety profile, compared to free DOX, suggesting that the use of polymeric nanocarriers would reduce the side effects of DOX. Moreover, the H&E and TUNEL assays were used to analyze the excised tumors to further evaluate the efficacy of the treatments. Figure 9 illustrates that in the case of DOX-loaded PMs, fewer tumor cells were observed, in comparison to the other formulations. Furthermore, the result of the TUNEL assays contributed to the elucidation of the therapeutic effect of the drugencapsulating micelles. Specifically, a marked increment in the number of apoptotic cells was observed following the used of DOX-loaded PMs, compared to the other two treatments. In this regards, the DOX-loaded PMs could be considered as a highly effective and safe formulation.

suggests that the release rate of DOX from PMs can be highly enhanced under cotriggered conditions. For physiological conditions (pH 7.4), the release of DOX was slower than that under triggered conditions. Cytotoxicity Test. The cytotoxicity of empty micelles and DOX-loaded micelles was assessed with human hepatocellular carcinoma HepG2 cells. Notably, the PMs showed remarkably lower cytotoxicity, with almost 100% cell viability, whereas DOX-loaded PMs were highly toxic at the concentration 30 μg/mL (Figure 6). This indicated that the empty PMs were nontoxic to HepG2 cells or suggested the good biocompatibility of this copolymer. Meanwhile, 30% of cells treated with DOX-loaded PMs remained, demonstrating the rapid release of DOX under the dual-triggered environment, as expected from the in vitro release profile of DOX-loaded PMs. Cellular Uptake. Confocal laser-scanning microscopy was used to analyze cell internalization and intracellular drug release of the PMs in comparison with free DOX in HepG2 cells (Figure 7). After 1 h of incubation, the free DOX sample showed a stronger fluorescence intensity than did the DOXloaded PMs, owing to the slower rate of energy-dependent endocytosis of the polymeric particles.23,24 After a longer incubation time, the DOX fluorescence intensity from DOXloaded PMs increased incredibly, which affirms the intracellularly triggered release of DOX from polymer micelles caused by an acidic environment or a high concentration of GSH. Therapeutic Efficacy of DOX-Loaded PMs. The antitumor efficacy of the DOX-loaded PMs was evaluated in vivo in HepG2 liver tumor-bearing mice. Different drug formulas, including free DOX and DOX-loaded PMs at the dose of DOX to be 2 mg per kg of mice body weight, were intravenously administrated, and normal saline was injected as control group. The tumor growth rate was recorded and compared among the different treatment groups. In the group treated with normal saline (control group), the tumor volume briskly increased, whereas in the group treated with the DOXcontaining formulations (Figure 8A), the tumor growth rates were effectively suppressed because of the intracellular inhibition activities of DOX.25,26 When comparing free DOX

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CONCLUSIONS The reported results show that a dual redox- and pH-sensitive amphiphilic copolymer was successfully synthesized, and it exhibited not only a sharp pH-dependence but also a good redox-responsive micellization−demicellization behavior. Under physiological conditions, this amphiphilic copolymer could form biocompatible micelles and encapsulate the hydrophobic anticancer drug, doxorubicin. The DOX-loaded PMs showed rapid stimuli-responsive release in both acidic environments and environments with a high concentration of reducing agent, as demonstrated in the drug release profile in vitro. Notably, in the animal model, DOX-loaded PMs had H

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(21) Chen, W.; Zhong, P.; Meng, F.; Cheng, R.; Deng, C.; Feijen, J.; Zhong, Zh. J. Controlled Release 2013, 169, 171−179. (22) Gue, M.; Yan, Y.; Zhang, H.; Yan, H.; Cao, Y.; Liu, K.; Wan, S.; Huang, J.; Yue, W. J. Mater. Chem. 2008, 18, 5104−5112. (23) Sun, C. Y.; Dou, S.; Du, J. Z.; Yang, X. Z.; Li, Y. P.; Wang, J. Adv. Healthcare Mater. 2013, 3, 261−272. (24) Yu, S.; Ding, J.; He, C.; Cao, Y.; Xu, W.; Chen, X. Adv. Healthcare Mater. 2014, 3, 752−760. (25) Markman, M. Expert Opin. Pharmacother. 2006, 7, 1469−1474. (26) Maluf, F. C.; D, M.; Spriggs, D. Gynecol. Oncol. 2002, 85, 18−31. (27) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 2039−2044. (28) Kabanov, A. V.; Nazarova, I. R.; Astafieva, I. V.; Batrakova, E. V.; Alakhov, V. Y.; Yaroslavov, A. A.; Kabanov, V. A. Macromolecules 1995, 28, 2303−2314.

enhanced therapeutic efficacy, compared to free DOX. These results suggested that this dual-sensitive copolymer might be promising as a potential smart nanocarrier for cancer therapy.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra of MPEG-COOH, NHS-activated MPEG, and MPEG-cystamine; GPC data of PAE-g-DSMPEG. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.5b00423.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +82 31 290 7267. Fax: +82 31 292 8790. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through a National Research Foundation of Korea grant funded by the Korean Government (MEST) (20100027955) and the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, & Future Planning (2012M3A9B6055205)

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DOI: 10.1021/acs.macromol.5b00423 Macromolecules XXXX, XXX, XXX−XXX