pH-Sensitive Docetaxel-Loaded d-α-Tocopheryl Polyethylene Glycol

Article pubs.acs.org/Biomac

pH-Sensitive Docetaxel-Loaded D‑α-Tocopheryl Polyethylene Glycol Succinate−Poly(β-amino ester) Copolymer Nanoparticles for Overcoming Multidrug Resistance Shuang Zhao,† Songwei Tan,† Yuanyuan Guo, Jing Huang, Min Chu, Hudan Liu, and Zhiping Zhang* Tongji School of Pharmacy & National Engineering Research Center for Nanomedicine, Huazhong University of Science and Technology, Wuhan 430030, China ABSTRACT: Multidrug resistance (MDR) is one of the major obstacles to successful chemotherapy. Overexpression of drug efflux transporters such as P-glycoprotein (P-gp) is an important factor responsible for MDR. Herein, a novel copolymer, D-α-tocopheryl polyethylene glycol 1000-block-poly(β-amino ester) (TPGS-b-PBAE, TP), was synthesized for overcoming multidrug resistance by the synergistic effect of the pH-sensitive behavior of PBAE and P-gp inhibiting activity of TPGS. Docetaxel (DTX) was chosen as the model drug. The resulting DTX-loaded nanoparticles were stable at pH 7.4, while they dissociated in a weakly acidic environment (pH 5.5) and released the incorporated DTX quickly. The DTX-loaded TP nanoparticles increased the cell cytotoxicity against both drug-sensitive human ovarian A2780 and drug-resistant A2780/T cells. The IC50 of DTX-loaded TP against A2780/T cells was 100-fold lower than that of commercial DTX. This was associated with enhanced DTX-induced apoptosis and cell arrest in the G2/M phase. Furthermore, P-gp inhibition assays, including enhancement of the fluorescence intensity of rhodamine 123 and reduction of the intracellular ATP levels, confirmed the P-gp inhibition nature of the TP copolymer. The use of the TP copolymer is a new approach to improve the therapeutic effect of anticancer drugs in MDR tumors.

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release in tumor cells.13−15 Among these pH-sensitive polymers, poly(β-amino ester) (PBAE), which was developed by Langer’s group via a Michael-type polymerization, is widely used as a biodegradable and low-cytotoxicity material in anticancer drug delivery systems.16−18 It has a basic character with a pKb value of about 6.5 due to its tertiary amine group. It protonates under the endosome pH value and facilitates the escape of encapsulated drugs/genes via the “proton sponge” effect. To date, many PBAEbased nanosized drug delivery systems have been developed and have shown improved cytotoxicity against tumor cells.19−23 D-α-Tocopheryl polyethylene glycol succinate (vitamin E TPGS, or simply TPGS) is a derivative of natural vitamin E (αtocopherol) and polyethylene glycol 1000 approved by the FDA, which is used as a solubilizer, an absorption enhancer, and a vehicle for drug delivery systems.24 Nanoparticles based on this material have exhibited enhanced drug encapsulation efficiency, cellular uptake, and cell cytotoxicity on cancer cells as well as an extended circulation time of up to 360 h, increased oral bioavailability of up to 78%, and enhanced therapeutic efficiency.25−28 More importantly, TPGS has been proved as an inhibitor of P-gp via inhibition of the activity of the ATPase part of P-gp; thus, it has shown great potential in overcoming MDR.29 Some TPGS-related copolymers, such as the PLA−TPGS

INTRODUCTION About one out of every four deaths is caused by cancer, and both the mortality and the incidence of cancer are still continuously rising every year. Cancer chemotherapy is still one of the most effective treatments, but successful chemotherapy is usually hampered by one main obstacle, multidrug resistance (MDR). MDR is a frequent phenomenon whereby cancer cells become resistant to the cytotoxic effects of various structurally and mechanically unrelated chemotherapeutic agents. The mechanisms of MDR are complex. Among them, overexpression of drug efflux pumps, such as P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporter, is one of the main causes of MDR. P-gp can dramatically reduce the intracellular drug concentration and thus limit the cytotoxic effects of drugs in tumors.1 To solve this problem, many drug delivery systems based on nanotechnology have been developed to increase the drug accumulation in tumors through introduction of stimulus-responsive burst release of entrapped drug from polymeric nanoparticles,2−5 codelivery of different functional anticancer agents into the same nanoparticle,6−8 coadministration of a P-gp inhibitor such as verapamil or siRNA along with an anticancer drug, and so on.9−12 As we know, the pH in tumor tissue and endosomes is more acidic (pH 6.0−5.0) compared to the physiological pH of 7.4. The physical properties of pH-responsive nanocarriers, such as swelling/shrinking and particle dissociation and aggregation, are responsive to environmental pH changes. In turn, these properties result in acidic pH-triggered rapid anticancer drug © 2013 American Chemical Society

Received: April 10, 2013 Revised: June 27, 2013 Published: July 1, 2013 2636

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Table 1. Synthesis and Properties of PBAE and TPGS-b-PBAE

a

polymer

feed molar ratio (TPGS-A:BDD:TPD)

yield (%)

product molar ratio (TPGS-A:BDD:TPD)a

TPGS conent (wt %)

pKbb

PBAE TP13 TP9

0:10:11 1:10:11 1:10:13

65.6 58.6 40.1

0:17:18 1:13:13 1:9:9

0 22.0 28.9

6.87 7.00 7.10

Determined by 1H NMR. bMeasured by the titration method. cooled to 0 °C, followed by addition of acryloyl chloride (0.56 g) dropwise. The mixture was stirred at 0 °C for 2 h, and then the temperature was raised to room temperature. After a further 24 h, the reaction mixture was purified by being washed with dilute HCl solution, followed by precipitation with hexane. TP copolymers were synthesized via a Michael-type step polymerization for 48 h at 50 °C in N,N-dimethylformamide using TPGS-A, BDD, and TDP as the monoacrylate, diacrylate ester, and diamine, respectively.20,21,33 At the end of the reaction, the solution was cooled to room temperature and precipitated in diethyl ether. The product was further washed three times to remove the unreacted monomers. The polymers with various molecular weights were prepared by adjusting the feed molar ratio of TPGS-A, BDD, and TDP (Table 1). 1 H NMR of TPGS-b-PBAE (CDCl3, ppm): 0.86 (12H, −CH(CH3)CH3 and −CH2CH(CH3)CH2−, TPGS), 2.50 (4H, −NCH2CH2COO−, PBAE), 2.59 (2H, PhCH2CH2−, TPGS), 2.66 (4H, −NCH2CH2COO−, PBAE), 2.80 (2H, −PhOCOCH2CH2COO−, TPGS), 2.87 (4H, −NCH 2 CH 2 CH−, PBAE), 2.95 (2H, −PhOCOCH2CH2COO−, TPGS), 3.65 (92H, −OCH2CH2O−, TPGS), 4.10 (4H, −COOCH2CH2−, PBAE), 4.25 (4H, −COOCH2CH2O−, TPGS), 1.89−2.09 (9H, CH3Ph, TPGS; 4H, −NCH2CH2CH−, PBAE), 1.00−1.80 (−CH2CH2CH2CH(CH3)CH2CH2CH2CH(CH3)CH2CH2CH2CH(CH3)CH3 and −OC(CH3)(CH2)−, TPGS; −COOCH2CH2 and −NCH2CH2CH(CH2)CH2, PBAE). Non-pH-sensitive copolymer TPGS-b-PLA (TL, Mn = 10 013) was synthesized as in our previous work.26 Preparation of Docetaxel-Loaded Nanoparticles. DTX-loaded nanoparticles (NPs) were fabricated by a modified solvent extraction/ evaporation method. DTX (0.6, 1.5, or 3.0 mg), 30 mg of TP/TL copolymer or PBAE, and 2 mg of the phospholipids DOPC and DSPE-PEG in a 9:1 molar ratio were codissolved in 1 mL of dichloromethane (denoted as TP/TL NPs and PBAE NPs, respectively). The organic phase was then dispersed into 6 mL of deionized water dropwise with ultrasonication in an ice bath for 3 min before evaporation overnight. After the collection and washing step by ultracentrifugation, the resulting particles were resuspended in phosphate-buffered saline (PBS) before use. Dil-loaded NPs were prepared by using Dil instead of DTX. Blank NPs were prepared in the same way without DTX in the feed. The drug loading efficiency of PBAE and TP NPs was determined by high-performance liquid chromatography (HPLC; Hitachi L-2000, equipped with a reversedphase Inertia ODS-3 C18 column (150 × 4.6 mm, pore size 5 μm, Agilent)). The mobile phase consisted of a mixture of water and acetonitrile (50:50, v/v). The elution rate was 1.0 mL/min, and the detection wavelength was set at 227 nm. The drug loading efficiency (DLE) was calculated as follows:

copolymer and a star-shaped copolymer of ditocopherol PEG 2000 succinate, have been developed and show the ability to overcome MDR.30,31 To the best of our knowledge, TPGS-based stimulus-responsive copolymers have not been reported, so in this study, a novel pHsensitive copolymer, TPGS-b-PBAE (TP), was synthesized to realize the synergistic effect of the pH-responsive release behavior and P-gp inhibiting effect to overcome MDR. At physiological pH (7.4), the copolymer behaves as an amphiphilic triblock copolymer with a small hydrophobic head (vitamin E), 15−20% PEG and 70−80% hydrophobic PBAE segments. Docetaxel (DTX), a P-gp substrate, was chosen as the model drug. It can be encapsulated in the PBAE core of the TP nanoparticle. The nanoparticles undergo a hydrophobic−hydrophilic transition under low pH within cancer cells, result in rapid intracellular drug release due to the protonation of PBAE, and realize “endosome escape”. Then the TPGS segment of TP copolymer binds with P-gp to prevent DTX from being pumped out. As a result, the cell cytotoxicity can be greatly improved, especially against MDR cells. In general, this work provides a simple but effective cancer chemotherapy.

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EXPERIMENTAL SECTION

Materials. TPGS was obtained from Sigma-Aldrich (St. Louis, MO). 1,4-Butanediol diacrylate (BDD; 99%), 4,4′-trimethylenedipiperidine (TDP; 97%), and acryloyl chloride (AC; 96%) were from Alfa Aesar, China. The lipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N(methoxypolyethylene glycol 2000) (DSPE-PEG) were purchased from Avanti Polar Lipids, Alabaster, AL. Docetaxel of purity 99% was obtained from Jinhe Ltd., China. Propidium iodide (PI), RNase A, and trypsin−EDTA were purchased from Sigma-Aldrich. RPMI-1640 medium was from Gibco BRL (Gaithersberg, MD). The lipophilic tracer Dil (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) was from Invitrogen, Carlsbad, CA. Penicillin−streptomycin, fetal bovine serum (FBS), and trypsin without EDTA were purchased from Hyclone, Waltham, MA. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide), rhodamine 123 (Rh123), and Hoechst 33342 were purchased from Biosharp, South Korea. All the solvents used were of analytical grade and were procured from Sinopharm, China. Human ovarian cancer cell line A2780, A2780/paclitaxel (A2780/T, multi-drug-resistant variant), and the annexin V− fluorescein isothiocyanate (FITC)/PI double staining assay kit were supplied by KeyGEN, China. Characterization. 1H NMR spectra were collected on a Bruker AVANCE III 400 MHz NMR spectrometer with CDCl3 as the solvent. Fourier transform infrared spectroscopy (FTIR) data were recorded on a Bruker VERTEX 70 FTIR spectrophotometer. Dynamic light scattering (DLS) data were obtained on a Brookhaven ZetaPlus particle size analyzer with a laser wavelength of 658 nm and at an angle of 90° at 25 °C. Scanning electron microscopy (SEM) images were obtained on a JEOL JSM-6490LA instrument operated at 5 kV. The sample was fixed on a stub as a thin film and coated with gold before the test. Ultraviolet− visible (UV−vis) absorption spectra were recorded on a Hitachi U-2900 UV−vis spectrophotometer at 550 nm. Synthesis of TPGS-b-PBAE Copolymers. TPGS acrylate (TPGS-A) was synthesized by TPGS and acryloyl chloride as reported elsewhere.19,32 Briefly, 3.03 g of TPGS was dissolved in 50 mL of anhydrous DCM with 0.5 mL of triethylamine. The solution was

DLE (%) =

weight of incorporated DTX in NPs × 100 feed amount of DTX in fabricating NPs

(1)

pH Sensitivity of Nanoparticles. To investigate the pH sensitivity property of the NPs, the light absorbance of the nanoparticle solutions at different pH values was tested. The wavelength was chosen as 550 nm because there is no absorption for both DTX and the polymers. In each case, the pH of the solution was first adjusted to 8.0. With the addition of 0.1 N HCl solution, the pH and absorbance were recorded. The particle size of the NPs was measured by DLS if an obvious absorbance change occurred. In Vitro Drug Release. The release profiles of DTX from DTXloaded NPs were studied using a dialysis tube (MWCO = 3500 Da) at 37 °C. To acquire the sink conditions, in vitro drug release tests were performed at a low drug concentration. Briefly, 5 mg of DTX-loaded 2637

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Thereafter, 5 μL of annexin V−FITC and 5 μL of PI were added, and the solution was mixed for 30 min in the dark. The stained cells were analyzed using a flow cytometer (Becton Dickinson, San Jose, CA). Cell Cycle Distribution Analysis. A2780/T cells were seeded onto six-well plates (1 × 105 cells/well) with DTX or DTX-loaded NPs (drug concentration of 0.5 μg/mL). After 24 h of incubation, the cells were washed twice with cold PBS and fixed overnight with 70% precooled alcohol at −20 °C. RNase A (100 μg/mL) was then added for 15 min at 37 °C followed by staining with PI solution (50 μg/mL) for 30 min in the dark. The DNA content was measured by the FACS Calibur system (Becton Dickinson), and the percentage of cells in each phase of the cell cycle was calculated using the ModFit software (Verity Software House, Topsham, ME). Intracellular ATP Level Assay. A2780/T cells were seeded onto 12-well plates (1 × 104 cells per well), incubated for 24 h at 37 °C, and then treated with 25 μg/mL TPGS and blank PBAE or TP NPs. Intracellular ATP levels were determined using the luciferin− luciferase-based ATP luminescence assay kit (Beyotime Institute of Biotechnology, China). Rhodamine 123 Efflux. Rh123, a P-gp substrate fluorescent dye, is an index of assaying the transport activity of P-gp. A2780/T cells were seeded onto six-well plates (1 × 105 cells/well) with 10 μg/mL TPGS or 50 μg/mL blank PBAE/TP NPs for 24 h. They were then incubated with 5 μg/mL Rh123 for 30 min at 37 °C. At the end of incubation, the cells were washed three times with PBS to remove free Rh123 and kept in dye-free medium. The fluorescence intensity of Rh123 in the cells was measured by fluorescence-activated cell sorting (FACS) using ModFit software. Western Blot Analysis. To determine the protein levels of P-gp, A2780/T cells were washed twice with ice-cold PBS and lysed in buffer (50 mM Tris−HCl, pH 8.0, 150 mM NaCl, 100 μg/mL PMSF (phenylmethanesulfonyl fluoride), 1% TritonX-100), then protease inhibitor cocktail (Sigma-Aldrich) was added for 30 min in an ice bath, and the solution was centrifuged at 12 000 rpm for 5 min. Equal amounts of protein extract (30 μg) were resolved on 6% SDS−PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% skim milk in TBST (10 mM Tris− HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h. The primary antibodies for P-gp (Calbiochem, San Diego, CA) and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) were diluted according to the manufacturer’s recommendations and the membranes incubated overnight at 4 °C. After washing with TBST, the membranes were further incubated for 2 h with horseradish peroxidase (HRP) and conjugated goat antimouse secondary antibody (Santa Cruz Biotechnology). Reactive protein was detected by ECL chemiluminescence system (Santa Cruz Biotechnology). Quantity One software was used to quantify the protein band intensities. Statistical Analysis. Each experiment was repeated at least three times. Numerical data are expressed as the mean ± standard deviation. The differences in the mean were analyzed by one-way analyis of variance (ANOVA) using SPSS software (version 19.0). The statistical significance was set as p < 0.05.

NPs was dispersed in 10 mL of the respective PBS buffer, allowed to stabilize for 30 min, and then placed in a dialysis tube. The dialysis tube was immersed in 50 mL of PBS solution (pH 7.4, 6.8, or 5.5) in a beaker and then placed in a 37 °C water bath shaker at 110 rpm. Samples were drawn at the desired time intervals, and the drug concentration was determined by HPLC as described above. Experiments for all samples were performed three times at each pH value. Cell Culture. A2780 and A2780/T cells were cultured in RPMI1640 medium supplemented with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere incubator with 5% CO2 at 37 °C. A2780/T cells were supplemented with 400 ng/mL paclitaxel. After the cells grew to 80−90% confluence, they were trypsinized with 0.125% trypsin−EDTA (diluted in fresh medium). In Vitro Cellular Uptake of Nanoparticles. Dil-loaded PBAE and TP NPs were used as a probe, and the cellular uptake was analyzed by confocal laser scanning microscopy (CLSM; Leica TCSNT1, Germany). A2780/T cells were separately seeded onto a 24-well plate, with coverslips over each well, at a density of 1.0 × 104 cells/well. After the cells reached 80% confluence, the medium was replaced with another medium containing Dil-loaded NPs at a concentration of 50 μg/mL at 37 °C for 2 h. After incubation, the wells were rinsed three times with cold PBS and then fixed using 4% paraformaldehyde for 15 min. The cells were further washed three times with 200 μL of PBS and stained with Hoechst 33342 for 8 min. The cells were then mounted on a glass slide for observation by CLSM. Cytotoxicity Assay in Drug-Sensitive A2780 Cells and Resistant A2780/T Cells. The in vitro cytotoxicity of DTX-loaded NPs and the clinical DTX formulation were determined by MTT assay. Briefly, A2780 and A2780/T cells in logarithmic growth were seeded onto 96-well plates at a seeding density of 5000 cells/well. Following overnight attachment, the culture medium in each well was carefully replaced with 100 μL of medium containing serial dilutions of the treatment samples, including the commercial docetaxel formulation, DTX-loaded PBAE NPs, and DTX-loaded TPs NPS. The concentration of DTX used in each group was 0.025, 0.25, 1.0, 2.5, 5, and 10 μg/mL, and the exposure duration was for 24, 48, and 72 h, respectively. At designated time intervals, 10 μL of MTT (5 mg/mL in PBS) was added to the wells. After incubation for 4 h, the culture solution was removed, leaving behind the precipitate. Thereafter, 150 μL of DMSO was added to each well to dissolve the formazan crystals while the plates were vigorously shaken using an automated shaker. The absorbance of each well was read on a microplate reader (Thermo Scientific, Pittsburgh, PA) at a test wavelength of 570 nm. All the experiments were done with seven parallel samples. The relative cell viability was calculated as the percentage of cell viability as compared with that of an untreated control. The IC50 (concentration resulting in 50% inhibition of cell growth) value was calculated by SPSS software (version 19.0). The experiment was repeated three times. Cell Apoptosis Analysis: Hoechst 33342 Staining. Nuclear morphologies of A2780/T cells with different treatments were determined by the Hoechst 33342 staining method. A2780/T cells were seeded onto a 24-well plate, with coverslips over each well, at a density of 1.0 × 104 cells per well followed by attachment for 24 h. The cells were then incubated with medium containing DTX or DTX-loaded NPs at identical DTX concentrations of 0.5 μg/mL. The control group was treated with drug-free culture medium. After incubation for 24 h, the wells were rinsed three times with cold PBS and then fixed with 200 μL of 4% paraformaldehyde for 15 min. The cells were further washed three times with 500 μL of PBS and stained with 200 μL of Hoechst 33342 (10 μg/mL) for 8 min. The cells were then mounted on a glass slide for observation by fluorescence microscopy (Olympus IX71, Tokyo, Japan). Cell Apoptosis Analysis: Annexin V−FITC/PI Double Staining. A2780/T cells seeded on six-well plates were treated with DTX and DTX-loaded PBAE, and DTX-loaded TP NPs (drug concentration of 0.5 μg/mL) at 37 °C for 24 h. Untreated cells were used as the control. At the end of the incubation period, the cells were trypsinized, collected, and resuspended in 200 μL of binding buffer.

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RESULTS AND DISCUSSION Synthesis and Characterization of TPGS-b-PBAE Copolymer. PBAE and TP copolymers were synthesized via Michael addition polymerization (Scheme 2). Two kinds of block copolymers were synthesized by changing the molar ratio of TPGS-A to BDD to TDP. The results are listed in Table 1. The chemical structures of TPGS-A, TP, and PBAE were confirmed by 1H NMR (Figure 1a) and FTIR (Figure 1b). As seen from the spectrum of TPGS-A, the signal at 3.65 ppm was characteristic of OCH2CH2− in the PEG units and the peak at 0.86 ppm was for the −CH3 protons of the long-chain alkyl group of TPGS. The three new peaks between 5.80 and 6.50 ppm were attributed to the −CHCH2 end group of TPGS-A. As compared to that of TPGS, the FTIR absorption at 1736 cm−1 for TPGS-A became stronger and a new peak at 1645 cm−1 appeared, 2638

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Table 2. Characteristic Properties of DTX-Loaded NPs

polymer

drug feeding concn (wt %)

PBAE PBAE PBAE TP13 TP9 TL

2 5 10 5 5 5

a

particle sizea (nm)

PDIa

± ± ± ± ± ±

0.124 0.119 0.139 0.142 0.140 0.134

257 261 265 247 228 236

19 16 10 8 7 12

ζ potential (mV) −25.5 −26.1 −25.8 −22.2 −20.1 −22.8

± ± ± ± ± ±

1.5 2.1 2.4 3.5 2.9 2.7

DLEb (%) 56.7 52.3 44.3 45.6 40.9 40.2

± ± ± ± ± ±

5.6 6.4 5.1 2.3 6.4 3.1

Measured by DLS. bMeasured by HPLC.

its maximum capacity.35 With introduction of TPGS in copolymer NPs, the DLE, particle size, and ζ potential were decreased compared with those of PBAE NPs. The decrease of the surface charge of copolymer NPs may be attributed to the charge shielding of the PEG segment in TPGS.36,37 The decreased particle size and DLE may be caused by the increased hydrophilicity of the copolymers.38 TL NPs were also prepared with a diameter and DLE close to those of TP NPs (Table 2). The surface morphology of TP13 NPs is seen from the inset in Scheme 1 with a spherical shape and diameter of about 200 nm in accordance with the dynamic light scattering results. pH Sensitivity of the Nanoparticles. To further understand the pH sensitivity of the blank NPs, the changes of the absorbance in response to an acidic environment were investigated in PBS at different pH values. As shown in Figure 2, Figure 1. 1H NMR (a) and FTIR (b) spectra of TPGS, TPGS-A, PBAE, and the TPGS-b-PBAE copolymer (TP9).

which belonged to the stretching vibration of the CO bond (νCO) and CC bond (νCC), respectively. This result proved the successful acrylate reaction between TPGA and acrylate chloride. For the TP copolymer, besides the characteristic peaks of TPGS at 3.65 and 0.86 ppm, a signal at 4.20 ppm, which was ascribed to the −OCOCH2− protons of the BDD units, was also found. Meanwhile, the signals at 2.85, 2.65, and 2.52 ppm were ascribed to −N(CH2)2−, −NCH2CH2OCO−, and −NCH2CH2OCO−, respectively.19,34 The TPGS content in the TP copolymer could be calculated on the basis of the peak area of the 3.65 and 4.20 ppm peaks. For the FTIR spectrum of the TP copolymer, the peak at 1387 cm−1 stood for the stretching vibration of the C−N bond (νC−N) in the PBAE block. The C−O stretching vibration (νC−O) of PEG showed a peak at 1104 cm−1, which was the typical signal of TPGS.33 These two obvious signals implied the synthesis of the TP copolymer. However, another new, strong peak at 1672 cm−1 occurred in the TP copolymer but not in PBAE. This might be attributed to the νCO of the PBAE segment, which was shifted to lower wavenumber from 1736 cm−1. This result indicated that a strong hydrogen-bonding interaction exists between the PBAE block and TPGS block. Characterization of Docetaxel-Loaded Nanoparticles. The particle size, ζ potential, and DLE of DTX-loaded NPs are described in Table 2. The DLE of PBAE NPs decreased from 56.7 ± 5.6% for 2% drug feeding to 52.3 ± 6.4% and 44.3 ± 5.1% for 5% and 10% drug feeding, respectively, with no significant difference in the particle size. This phenomenon was in accordance with previously reported PEG−PBAE micelles.21 It may be caused by the limited capacity of the polymer to encapsulate the specific drug, and more drug might be wasted during the nanoparticle fabrication process when it is beyond

Figure 2. pH-dependent absorbance (a) and particle size distribution profile (b) of blank PBAE, TP13 NPs, TP9 NPs, and TL NPs.

the TP NPs and PBAE NPs exhibited high stability at the psychological pH of 7.4. However, they presented a sharp pH-sensitive protonated/unprotonated transition in the tumor microenvironment (pH transition between 6.6 and 6.0). The tertiary diamine moieties of PBAE were gradually ionized, and thus, the NPs were gradually broken at a pH of 5.5−6.5, resulting in a sharp decrease in both the solution absorbance and particle size. A slight increase in absorbance in TP NPs was observed at a pH range of 7.1−6.7. It may be caused by the increase of particle size due to the slight protonation of the inner PBAE core in NPs. For PBAE NPs, a similar behavior occurred at pH 6.7−6.4. The different pH-responsive behavior of TP and PBAE NPs could be attributed to the chain length of the PBAE segment. The PBAE segments with a high molecular weight may be difficult to ionize due to the entropic effect.19 For non-pH-sensitive TL NPs, both the absorbance and particle size remained the same at different pH values. 2639

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Scheme 1. Scheme of Drug-Loaded and pH-Dependent Release from TPGS-b-PBAE NPs

Figure 3. In vitro drug release profiles from DTX-loaded NPs in pH 7.4 (solid lines), 6.8 (dotted lines), and 5.5 (dashed lines) PBS solutions.

Figure 4. CLSM images of Dil-loaded PBAE NPs (a), TP13 NPs (b), and TP9 NPs (c) in A2780/T cells after 2 h of incubation.

pH-Triggered Drug Release. The release behavior of DTX from the NPs was tested under different pH conditions, pH 7.4 (corresponds to the environment of blood), pH 6.8 (the pH of tumor tissue), and pH 5.5 (simulates the pH in mature endosomes of tumor cells). As shown in Figure 3, PBAE NPs and TP NPs exhibited obvious pH-related release behavior. Less than 25% of the incorporated DTX was released from the NPs within 12 h in PBS (pH 7.4). At pH 6.8, the drug release ratio was a little higher than that at pH 7.4; about 35% loaded

drug was released due to the slight protonation of the PBAE segment. However, a rapid release occurred at pH 5.5, and more than 65% of entrapped DTX was released after 12 h of incubation, which may be attributed to the acidic pH-induced deformation of the core−shell structure of the NPs. For nonpH-sensitive TL NPs, the drug release behavior was almost the same at pH 7.4 and 6.8, but it was accelerated at pH 5.5 due to fast hydrolysis of the PLA segment. The in vitro drug release results indicated that the PBAE copolymer NPs could realize 2640

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Scheme 2. Synthetic Scheme of the TPGS-b-PBAE Copolymer

Figure 5. In vitro cytotoxicity of DTX-loaded NPs and DTX against A2780 (a) and A2780/T (b) cancer cells after treatment for 24, 48, and 72 h. Drug resistance index (RI) (c) and drug resistance reverse index (RRI) (d). RI is the ratio of the IC50 of MDR cells to that of sensitive cells for the same formulations, RI = IC50(A2780/T)/IC50(A2780). RRI is the ratio of the IC50 of the DTX solution to that of NP formulations, RRI = IC50(DTX)/IC50(DTX-loaded NPs). Key: *, p < 0.05; **, p < 0.01; 1, NPs vs DTX, p < 0.05; 2, TP13 NPs vs PBAE NPs, p < 0.05; 3, TP9 NPs vs PBAE NPs and TP13 NPs, p < 0.05.

Cellular Uptake of Dil-Loaded Nanoparticles. To test the ability of the cellular uptake of NPs, Dil, a widely used

burst release of entrapped drug after being taken up by tumor cells and result in targeted cytotoxicity of tumors.39 2641

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Figure 6. Nucleus apoptosis assay of A2780/T cells treated with the control (a), DTX (b), DTX-loaded PBAE NPs (c), DTX-loaded TP13 NPs (d), and DTX-loaded TP9 NPs (e) for 24 h.

Figure 7. Cell apoptosis analysis of A2780/T cells by flow cytometry using staining of annexin V−FITC and PI treated with the control (a), DTX (b), DTX-loaded PBAE NPs (c), DTX-loaded TP13 NPs (d), and DTX-loaded TP9 NPs (e) for 24 h.

replacement fluorescent marker of hydrophobic drug, was loaded into NPs to monitor the NPs and Hoechst 33342 used in the staining of the nucleus. The cellular uptake was performed by CLSM in the A2780/T cell line. As shown in Figure 4, A2780/T cells were incubated with Dil-loaded PBAE NPs, TP13 NPs, and TP9 NPs for 2 h, respectively. After 2 h of exposure, all samples exhibited significant red fluorescence throughout the cytoplasm, which is closely located around the nuclei (blue). This suggested that the NPs had been effectively taken up by the cells as it is hard for Dil to diffuse into the cytoplasm separately. Only the uptake of Dil-loaded NPs could

achieve the significant distribution in the cytoplasm.40 Furthermore, TP NPs exhibited significantly higher cell uptake compared to PBAE NPs; especially TP9 NPs demonstrated enhanced cell uptake compared to TP13 NPs as seen from Figure 4. This indicated that TPGS could effectively facilitate drug accumulation in cancer cells, which may be attributed to the mediation by TPGS as a permeation enhancer.4 In Vitro Cytotoxicity of DTX-Loaded Nanoparticles against A2780 and A2780/T Cells. To determine the cytotoxic activities of DTX-loaded NPs, in vitro cell cytotoxicity experiments were performed in both A2780 and drug-resistant 2642

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Figure 8. Changes of cell cycle distribution in A2780/T cells treated with various formulations of the control (a), DTX (b), DTX-loaded PBAE NPs (c), DTX-loaded TP13 NPs (d), and DTX-loaded TP9 NPs (e) for 24 h.

PBAE NPs, 47.8 ± 6.1 for TP13 NPs, and 104.0 ± 13.0 for TP9 NPs. It was noteworthy that both TP13 and TP9 NPs exhibited a stronger ability in reversing drug resistance, as confirmed by the higher RRI and the lower RI with the treatment time ranges.37 Moreover, TP9 NPs more effectively reversed MDR than TP13 NPs. This might come from its higher TPGS content. In summary, both PBAE and TP NPs could promote DTX accumulation in A2780 and A2780/T cells, and thus, better treatment efficiency was achieved. The cytotoxicity of PBAE and TP NPs against drug-sensitive A2780 cells did not show much difference. The cell cytotoxicity may be mainly attributed to the fast DTX release and endosome escape via the “proton sponge” effect of PBAE in the cells.3,41−43 However, for drug-resistant A2780/T cells, where P-gp was overexpressed, DTX-loaded TP NPs mediated more effective anticancer activity than PBAE NPs. This may be caused by the synergistic effects of the TPGS and PBAE. Compared with PBAE NPs, the TPGS segment in the TP copolymer could weaken the function of the drug efflux pump, P-gp, after escape from the endosome. This would be helpful to keep a high intracellular DTX concentration, and the liberated DTX in the cytoplasm of the cells could exert its therapeutic effect. Therefor, the higher TPGS content in the polymer, the stronger the cytotoxicity. TPGS is a positive input in the treatment of MDR solid tumors. Cell Apoptosis Qualitative Assays: Fluorescent Morphological Study. It has been reported that drug-loaded NPs are able to induce cell death via the apoptosis pathway subsequent to microtubule disruption.44 In this study, Hoechst staining of nuclei was observed after a series of treatments. As shown in Figure 6, the nuclei of the control A2780/T cells were homogeneously stained in a manner similar to that of the cytoplasm (Figure 6a). Chromatin condensation and apoptotic body formation could be observed in the cells treated with DTX (Figure 6b), PBAE NPs (Figure 6c), TP13 NPs (Figure 6d), and TP9 NPs (Figure 6e), which were typical apoptotic features in A2780/T cells. The drug-loaded NPs induced more cell apoptosis than free DTX. In addition, the nucleus of the cells

A2780/T cells. As shown in Figure 5a,b, DTX exhibited significant cell cytotoxicity dependent on the incubation concentration and exposure duration against drug-sensitive A2780 cells, but for drug-resistant A2780/T cells, this tendency was not observed. Even after incubation for 72 h with a DTX concentration as high as 10 μg/mL, the cell viability of A2780/ T cells was still about 40%. The cytotoxicity of DTX against A2780/T cells was not good due to their drug-resistant nature. However, the DTX-loaded NPs exhibited higher cell cytotoxicity against both types of cells than DTX and showed an obvious concentration-dependent tendency. DTX-loaded TP NPs were usually more effective than PBAE NPs in killing tumor cells, especially in drug-resistant A2780/T cells. IC50 was further calculated by curve fitting and is presented in the inset tables of Figure 5a,b. After incubation of the samples with A2780 cells for 24 h, the IC50 values became 0.270 ± 0.034 μg/mL for PBAE NPs, 0.220 ± 0.042 μg/mL for TP13 NPs, and 0.140 ± 0.012 μg/mL for TP9 NPs, which were significant decreases as compared with that of DTX (3.95 ± 0.563 μg/mL). Notably, after drug treatment for 72 h, the IC50 of PBAE, TP13, and TP9 NPs decreased more than 9-, 15-, and 22-fold against that of free DTX, respectively. As for A2780/T cells, which overexpress P-gp and exhibit the drug-resistant phenotype, the IC50 value of free DTX was much higher (17.68 ± 4.38 μg/mL) compared with those of DTX-loaded NPs (0.820 ± 0.106 μg/mL for PBAE, 0.370 ± 0.043 μg/mL for TP13, and 0.170 ± 0.034 μg/mL for TP9) after treatment for 24 h, which were more than 20-, 50-, and 100-fold decreases. The existence of TPGS would greatly enhance the treatment efficiency of the DTX-loaded NPs. To further present the statistically significant differences in inhibition rates and effects on the resistant cells among the various formulations, the concepts of the resistance index (RI; Figure 5c) and resistance reversion index (RRI; Figure 5d) were introduced. The RI decreased from 4.47 ± 0.22 for DTX to 3.03 ± 0.31 for PBAE NPs, 1.68 ± 0.21 for P-T20 NPs, and 1.21 ± 0.20 for TP9 NPs. The RRI also increased to 21.6 ± 5.0 for 2643

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proved the ability of the TP copolymer to overcome MDR in A2780/T tumor cells. Cell Cycle Arrest in A2780/T Cells. To further determine the apoptotic mechanism induced by the DTX-loaded NPs, the cell cycle distribution was analyzed. It has been proved that DTX is connected with mitosis inhibition and cell arrest in the G2/M phase and the increased G2/M phase arrest is a signal of cell growth restraint and cell division inhibition.11 According to Figure 8, the percentage of cells in the G2/M phase increased to 21.20%, 51.20%, 72.90%, and 78.22% for DTX and DTXloaded PBAE, TP13, and TP9 NPs, respectively, which were remarkably different from that of the control (14.05%) after 24 h of exposure. It is notably indicated that the NPs, especially TP NPs, presented a distinguished ability to induce cell cycle arrest in the G2/M phase, which was consistent with that of the cell apoptosis experiments. P-gp Inhibition Assays. It is well-known that P-gp is overexpressed on the membrane of A2780/T cells, which is one of the main causes of the MDR phenotype, and the drug efflux by P-gp is ATP-dependent.45 The P-gp expression level of A2780 and A2780/T cells used in this work was detected by Western blot (Figure 9c). Significant P-gp overexpression in A2780/T cells compared with A2780 cells was confirmed. The Rh123 retention fluorescent intensity and intracellular ATP levels were used to evaluate the effect of TP on P-gp and to explore the relevant regulating mechanism. As seen in Figure 9a, the fluorescence intensity of Rh123 was increased in A2780/T cells treated with the NPs or TPGS in comparison with the untreated cells, which indicated that the function of the P-gp pump was weakened. The increase of the TPGS content seemed to be able to strengthen the fluorescence intensity and improve the retention ability slightly. For ATPase assay (Figure 9b), free TPGS could be responsible for the sharp decrease of the intracellular ATP level (43 ± 3%). Compared to PBAE NPs (79 ± 4%), the TP NPs exhibited a stronger inhibition of the intracellular ATP level (44 ± 2% for TP13 NPs and 47 ± 4% for TP9 NPs), close to that of free TPGS. It is known that TPGS could bind with the ATPase part of P-gp, so the decrease in the consumption of ATP corresponded to the ability of the TP copolymer to reduce the activity of ATPase.46 Therefore, it might be concluded that the TPGS segment in the TP copolymer may inhibit the functions of P-gp, prevent the pump-out of drug, and thus result in an increase of the chemosensitivity of A2780/T cells. It was also found that PBAE could enhance the Rh123 retention ability and lower the ATP level slightly. A possible reason was that PBAE could interact with the cell membrane, like Pluronic29 and PEG−PLA,47 so as to change the activity of P-gp. This needs further investigation.

Figure 9. Rh123 intracellular accumulation in A2780/T cells that were untreated (control) or treated with TPGS (serving as a positive control) and blank copolymer NPs after 24 h of incubation (a), intracellular ATP levels in A2780/T cells treated with blank copolymer NPs after 24 h of incubation (b), and the expressions of P-gp protein in A2780 and A2780/T cells (c). An asterisk indicates p < 0.05 compared with the control group.

treated with TP NPs split more than those treated with PBAE formation, and TP9 NPs exhibited the highest significant effect on apoptosis. All the above results were in harmony with the results of the MTT assay. Cell Apoptosis Quantitative Analysis: Annexin−PI Staining Assay. To verify the MDR cell apoptosis rate induced by the DTX-loaded NPs, an annexin−PI staining assay was performed to carry out a quantitative analysis of the early apoptosis (Figure 7). The lower right quadrant (annexin V-positive and PI-negative) is a symbol of the percentage of early apoptosis. The percentage of early apoptosis of A2780/T cells was 4.41% under controlled conditions, while after 24 h of incubation with free DTX and DTX-loaded PBAE NPs, TP13 NPs, and TP9 NPs, cell apoptosis was increased to 14.37%, 18.21%, 35.78%, and 39.15%, respectively. This was consistent with the results of cell apoptosis qualitative assays in the last section. Both the quantitative and qualitative results demonstrated that the DTX-loaded NPs significantly enhanced DTX-induced apoptosis. The apoptotic rates of cells treated with the TP NPs were significantly higher than those of any other treatments. This also

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CONCLUSION In summary, a novel dual-function pH-sensitive copolymer, TPGS-b-PBAE, was successfully synthesized and applied in anticancer drug delivery to overcome MDR. The copolymer solubilized the model hydrophobic drug DTX effectively, and the resulting drug-loaded NPs demonstrated a well-controlled pH-sensitive drug release behavior. Moreover, the TPGS segment of the copolymer was helpful to enhance the cellular accumulation and cell cytotoxicity of the DTX against drug-sensitive and drugresistant human ovarian cells. More importantly, the drug-loaded NPs showed great ability in overcoming multidrug resistance of tumor due to the synergistic effect of PBAE-induced pH-sensitive intracellular burst release and TPGS-mediated P-gp inhibition. We 2644

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survivin-targeting RNA with reduction-responsible cationic poly(βamino esters). Biomaterials 2012, 33 (27), 6495−6506. (13) Gao, W.; Chan, J. M.; Farokhzad, O. C. pH-responsive nanoparticles for drug delivery. Mol. Pharmaceutics 2010, 7 (6), 1913− 1920. (14) Chen, J.; Xing, M. M. Q.; Zhong, W. Degradable micelles based on hydrolytically degradable amphiphilic graft copolymers for doxorubicin delivery. Polymer 2011, 52 (4), 933−941. (15) Wu, X. L.; Kim, J. H.; Koo, H.; Bae, S. M.; Shin, H.; Kim, M. S.; Lee, B.-H.; Park, R.-W.; Kim, I.-S.; Choi, K.; Kwon, I. C.; Kim, K.; Lee, D. S. Tumor-targeting peptide conjugated pH-responsive micelles as a potential drug carrier for cancer therapy. Bioconjugate Chem. 2010, 21 (2), 208−213. (16) Lynn, D. M.; Langer, R. Degradable poly(β-amino esters): Synthesis, characterization, and self-assembly with plasmid DNA. J. Am. Chem. Soc. 2000, 122 (44), 10761−10768. (17) Shenoy, D.; Little, S.; Langer, R.; Amiji, M. Poly(ethylene oxide)-modified poly(β-amino ester) nanoparticles as a pH-sensitive system for tumor-targeted delivery of hydrophobic drugs: Part 2. In vivo distribution and tumor localization studies. Pharm. Res. 2005, 22 (12), 2107−2114. (18) Brito, L.; Little, S.; Langer, R.; Amiji, M. Poly(β-amino ester) and cationic phospholipid-based lipopolyplexes for gene delivery and transfection in human aortic endothelial and smooth muscle cells. Biomacromolecules 2008, 9 (4), 1179−1187. (19) Zhang, C. Y.; Yang, Y. Q.; Huang, T. X.; Zhao, B.; Guo, X. D.; Wang, J. F.; Zhang, L. J. Self-assembled pH-responsive MPEG-b(PLA-co-PAE) block copolymer micelles for anticancer drug delivery. Biomaterials 2012, 33 (26), 6273−6283. (20) Ko, J.; Park, K.; Kim, Y.-S.; Kim, M. S.; Han, J. K.; Kim, K.; Park, R.-W.; Kim, I.-S.; Song, H. K.; Lee, D. S.; Kwon, I. C. Tumoral acidic extracellular pH targeting of pH-responsive MPEG-poly(β-amino ester) block copolymer micelles for cancer therapy. J. Controlled Release 2007, 123 (2), 109−115. (21) Min, K. H.; Kim, J.-H.; Bae, S. M.; Shin, H.; Kim, M. S.; Park, S.; Lee, H.; Park, R.-W.; Kim, I.-S.; Kim, K.; Kwon, I. C.; Jeong, S. Y.; Lee, D. S. Tumoral acidic pH-responsive MPEG-poly(β-amino ester) polymeric micelles for cancer targeting therapy. J. Controlled Release 2010, 144 (2), 259−266. (22) Kim, M. S.; Gao, G. H.; Kang, S. W.; Lee, D. S. Evaluation of pH-sensitive poly(β-amino ester)-graft-poly(ethylene glycol) and its usefulness as a pH-sensor and protein carrier. Macromol. Biosci. 2011, 11 (7), 946−951. (23) Chen, Y. C.; Bathula, S. R.; Li, J.; Huang, L. Multifunctional nanoparticles delivering small interfering RNA and doxorubicin overcome drug resistance in cancer. J. Biol. Chem. 2010, 285 (29), 22639−22650. (24) Zhang, Z.; Tan, S.; Feng, S. S. Vitamin E TPGS as a molecular biomaterial for drug delivery. Biomaterials 2012, 33 (19), 4889−4906. (25) Feng, S. S.; Zhao, L.; Zhang, Z.; Bhakta, G.; Yin Win, K.; Dong, Y.; Chien, S. Chemotherapeutic engineering: Vitamin E TPGSemulsified nanoparticles of biodegradable polymers realized sustainable paclitaxel chemotherapy for 168h in vivo. Chem. Eng. Sci. 2007, 62 (23), 6641−6648. (26) Zhang, Z.; Lee, S. H.; Gan, C. W.; Feng, S. S. In vitro and in vivo investigation on PLA-TPGS nanoparticles for controlled and sustained small molecule chemotherapy. Pharm. Res. 2008, 25 (8), 1925−1935. (27) Huang, L.; Chen, H.; Zheng, Y.; Song, X.; Liu, R.; Liu, K.; Zeng, X.; Mei, L. Nanoformulation of D-α-tocopheryl polyethylene glycol 1000 succinate-b-poly(ε-caprolactone-ran-glycolide) diblock copolymer for breast cancer therapy. Integr. Biol. 2011, 3 (10), 993−1002. (28) Feng, S. S.; Mei, L.; Anitha, P.; Gan, C. W.; Zhou, W. Poly(lactide)-vitamin E derivative/montmorillonite nanoparticle formulations for the oral delivery of docetaxel. Biomaterials 2009, 30 (19), 3297−3306. (29) Collnot, E. M.; Baldes, C.; Schaefer, U. F.; Edgar, K. J.; Wempe, M. F.; Lehr, C. M. Vitamin E TPGS P-glycoprotein inhibition mechanism: Influence on conformational flexibility, intracellular ATP

believe that this material provides a new platform for treating MDR solid tumors.

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AUTHOR INFORMATION

Corresponding Author

*Fax and phone: +86-27-83601832. E-mail: zhipingzhang@ mail.hust.edu.cn. Author Contributions †

S. Zhao and S.W. Tan contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Basic Research Program of China (973 Program, Grant no. 2012CB932500), National Natural Science Foundation of China (NSFC) (Grant nos. 21204024 and 81241103), Doctoral Fund of the Ministry of Education of China (Grant no. 20120142120093), and Innovative Research Fund. We thank Professor Gao Li for his helpful advice and master degree candidate Zhaojing Wang for assistance with SDS−PAGE.

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REFERENCES

(1) Gottesman, M. M.; Fojo, T.; Bates, S. E. Multidrug resistance in cancer: Role of ATP-dependent transporters. Nat. Rev. Cancer 2002, 2 (1), 48−58. (2) Fleige, E.; Quadir, M. A.; Haag, R. Stimuli-responsive polymeric nanocarriers for the controlled transport of active compounds: Concepts and applications. Adv. Drug Delivery Rev. 2012, 64 (9), 866−884. (3) Meng, F.; Hennink, W. E.; Zhong, Z. Reduction-sensitive polymers and bioconjugates for biomedical applications. Biomaterials 2009, 30 (12), 2180−2198. (4) Gao, G. H.; Lee, J. W.; Nguyen, M. K.; Im, G. H.; Yang, J.; Heo, H.; Jeon, P.; Park, T. G.; Lee, J. H.; Lee, D. S. pH-responsive polymeric micelle based on PEG-poly(β-amino ester)/(amido amine) as intelligent vehicle for magnetic resonance imaging in detection of cerebral ischemic area. J. Controlled Release 2011, 155 (1), 11−17. (5) Ke, C. J.; Chiang, W. L.; Liao, Z. X.; Chen, H. L.; Lai, P. S.; Sun, J. S.; Sung, H. W. Real-time visualization of pH-responsive PLGA hollow particles containing a gas-generating agent targeted for acidic organelles for overcoming multi-drug resistance. Biomaterials 2013, 34 (1), 1−10. (6) Gao, Z.; Zhang, L.; Sun, Y. Nanotechnology applied to overcome tumor drug resistance. J. Controlled Release 2012, 45−55. (7) Ganta, S.; Amiji, M. Coadministration of paclitaxel and curcumin in nanoemulsion formulations to overcome multidrug resistance in tumor cells. Mol. Pharmaceutics 2009, 6 (3), 928−939. (8) Tan, S.; Li, X.; Guo, Y.; Zhang, Z. Lipid-enveloped hybrid nanoparticles for drug delivery. Nanoscale 2013, 5 (3), 860−872. (9) Hu, Q.; Li, W.; Hu, X.; Shen, J.; Jin, X.; Zhou, J.; Tang, G.; Chu, P. K. Synergistic treatment of ovarian cancer by co-delivery of survivin shRNA and paclitaxel via supramolecular micellar assembly. Biomaterials 2012, 33 (27), 6580−6591. (10) Wang, F.; Zhang, D.; Zhang, Q.; Chen, Y.; Zheng, D.; Hao, L.; Duan, C.; Jia, L.; Liu, G.; Liu, Y. Synergistic effect of folate-mediated targeting and verapamil-mediated P-gp inhibition with paclitaxelpolymer micelles to overcome multi-drug resistance. Biomaterials 2011, 32 (35), 9444−9456. (11) Shen, J.; Yin, Q.; Chen, L.; Zhang, Z.; Li, Y. Co-delivery of paclitaxel and survivin shRNA by pluronic P85-PEI/TPGS complex nanoparticles to overcome drug resistance in lung cancer. Biomaterials 2012, 33 (33), 8613−8624. (12) Yin, Q.; Shen, J.; Chen, L.; Zhang, Z.; Gu, W.; Li, Y. Overcoming multidrug resistance by co-delivery of Mdr-1 and 2645

dx.doi.org/10.1021/bm4005113 | Biomacromolecules 2013, 14, 2636−2646

Biomacromolecules

Article

synthetic α-aminoxy peptidomimetic. Int. J. Pharm. 2012, 424 (1), 33− 39. (47) Xiao, L.; Xiong, X. Q.; Sun, X. H.; Zhu, Y. H.; Yang, H.; Chen, H. B.; Gan, L.; Xu, H. B.; Yang, X. L. Role of cellular uptake in the reversal of multidrug resistance by PEG-b-PLA polymeric micelles. Biomaterials 2011, 32 (22), 5148−5157.

levels, and role of time and site of access. Mol. Pharmaceutics 2010, 7 (3), 642−651. (30) Li, P. Y.; Lai, P. S.; Hung, W. C.; Syu, W. J. Poly(L-lactide)vitamin E TPGS nanoparticles enhanced the cytotoxicity of doxorubicin in drug-resistant MCF-7 breast cancer cells. Biomacromolecules 2010, 11 (10), 2576−2582. (31) Wang, J.; Sun, J.; Chen, Q.; Gao, Y.; Li, L.; Li, H.; Leng, D.; Wang, Y.; Sun, Y.; Jing, Y. Star-shape copolymer of lysine-linked ditocopherol polyethylene glycol 2000 succinate for doxorubicin delivery with reversal of multidrug resistance. Biomaterials 2012, 33 (28), 6877−6888. (32) Kim, M. S.; Hwang, S. J.; Han, J. K.; Choi, E. K.; Park, H. J.; Kim, J. S.; Lee, D. S. pH-responsive PEG-poly(β-amino ester) block copolymer micelles with a sharp transition. Macromol. Rapid Commun. 2006, 27 (6), 447−451. (33) Huynh, C. T.; Nguyen, M. K.; Jeong, I. K.; Kim, S. W.; Lee, D. S. Synthesis, characteristics and potential application of poly(β-amino ester urethane)-based multiblock co-polymers as an injectable, biodegradable and pH/temperature-sensitive hydrogel system. J. Biomater. Sci., Polym. Ed. 2012, 23 (8), 1091−1106. (34) Gu, J.; Wang, X.; Jiang, X.; Chen, Y.; Chen, L.; Fang, X.; Sha, X. Self-assembled carboxymethyl poly (L-histidine) coated poly (β-amino ester)/DNA complexes for gene transfection. Biomaterials 2012, 33 (2), 644−658. (35) Dong, Y.; Feng, S.-S. Methoxy poly(ethylene glycol)-poly(lactide) (MPEG-PLA) nanoparticles for controlled delivery of anticancer drugs. Biomaterials 2004, 25 (14), 2843−2849. (36) Fang, C.; Kievit, F. M.; Cho, Y.-C.; Mok, H.; Press, O. W.; Zhang, M. Effect of cationic side-chains on intracellular delivery and cytotoxicity of pH sensitive polymer-doxorubicin nanocarriers. Nanoscale 2012, 4 (22), 7012−7020. (37) Jin, Y.-H.; Hu, H.-Y.; Qiao, M.-X.; Zhu, J.; Qi, J.-W.; Hu, C.-J.; Zhang, Q.; Chen, D.-W. pH-sensitive chitosan-derived nanoparticles as doxorubicin carriers for effective anti-tumor activity: Preparation and in vitro evaluation. Colloids Surf., B 2012, 94, 184−191. (38) Li, Z.; Hu, S.; Wang, J.; Cai, J.; Xiao, L.; Yu, L.; Wang, Z. MiR27a modulates MDR1/P-glycoprotein expression by targeting HIPK2 in human ovarian cancer cells. Gynecol. Oncol. 2010, 119 (1), 125− 130. (39) Chen, J.; Qiu, X.; Ouyang, J.; Kong, J.; Zhong, W.; Xing, M. M. Q. pH and reduction dual-sensitive copolymeric micelles for intracellular doxorubicin delivery. Biomacromolecules 2011, 12 (10), 3601−3611. (40) Su, X.; Fricke, J.; Kavanagh, D. G.; Irvine, D. J. In vitro and in vivo mRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles. Mol. Pharmaceutics 2011, 8 (3), 774−787. (41) Akash, M. S. H.; Rehman, K.; Li, N.; Gao, J. Q.; Sun, H.; Chen, S. Sustained delivery of IL-1Ra from pluronic F127-based thermosensitive gel prolongs its therapeutic potentials. Pharm. Res. 2012, 1−11. (42) Aryal, S.; Hu, C. M. J.; Zhang, L. Polymer−cisplatin conjugate nanoparticles for acid-responsive drug delivery. ACS Nano 2009, 4 (1), 251−258. (43) Sonaje, K.; Lin, K. J.; Wey, S. P.; Lin, C. K.; Yeh, T. H.; Nguyen, H. N.; Hsu, C. W.; Yen, T. C.; Juang, J. H.; Sung, H. W. Biodistribution, pharmacodynamics and pharmacokinetics of insulin analogues in a rat model: Oral delivery using pH-responsive nanoparticles vs. subcutaneous injection. Biomaterials 2010, 31 (26), 6849−6858. (44) Guan, M.; Zhou, Y.; Zhu, Q.-L.; Liu, Y.; Bei, Y.-Y.; Zhang, X.-N.; Zhang, Q. N-trimethyl chitosan nanoparticle-encapsulated lactosylnorcantharidin for liver cancer therapy with high targeting efficacy. Nanomedicine: Nanotechnol., Biol. Med. 2012, 8 (7), 1172−1181. (45) He, Q.; Gao, Y.; Zhang, L.; Zhang, Z.; Gao, F.; Ji, X.; Li, Y.; Shi, J. A pH-responsive mesoporous silica nanoparticles-based multi-drug delivery system for overcoming multi-drug resistance. Biomaterials 2011, 32 (30), 7711−7720. (46) Ma, B.; Chai, S.; Li, N.; To, K. K.; Kan, W. L. T.; Yang, D.; Lin, G. Reversal of P-glycoprotein-mediated multidrug resistance by a 2646

dx.doi.org/10.1021/bm4005113 | Biomacromolecules 2013, 14, 2636−2646