Novel Free-Paclitaxel-Loaded Redox-Responsive Nanoparticles

Sep 10, 2014 - PEG-SS-PTX/PTX NPs were relatively stable under normal conditions but disassembled quickly under reductive conditions, as indicated by ...
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Novel Free-Paclitaxel-Loaded Redox-Responsive Nanoparticles Based on a Disulfide-Linked Poly(ethylene glycol)−Drug Conjugate for Intracellular Drug Delivery: Synthesis, Characterization, and Antitumor Activity in Vitro and in Vivo Xingxing Chuan, Qin Song, Jialiang Lin, Xianhui Chen, Hua Zhang, Wenbing Dai, Bing He, Xueqing Wang,* and Qiang Zhang State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China S Supporting Information *

ABSTRACT: To address the obstacles facing cancer chemotherapeutics, including toxicity, side effects, water insolubility, and lack of tumor selectivity, a novel stimuli-responsive drugdelivery system was developed based on paclitaxel-loaded poly(ethylene glycol)-disulfide-paclitaxel conjugate nanoparticles (PEG-SS-PTX/PTX NPs). The formulation emphasizes several benefits, including polymer−drug conjugates/prodrugs, self-assembled NPs, high drug content, redox responsiveness, and programmed drug release. The PTX-loaded, selfassembled NPs, with a uniform size of 103 nm, characterized by DLS, TEM, XRD, DSC, and 1H NMR, exhibited excellent drug-loading capacity (15.7%) and entrapment efficiency (93.3%). PEG-SS-PTX/PTX NPs were relatively stable under normal conditions but disassembled quickly under reductive conditions, as indicated by their triggered-aggregation phenomena and drug-release profile in the presence of dithiothreitol (DTT), a reducing agent. Additionally, by taking advantage of the difference in the drug-release rates between physically loaded and chemically conjugated drugs, a programmed drug-release phenomenon was observed, which was attributed to a higher concentration and longer action time of the drugs. The influence of PEG-SSPTX/PTX NPs on in vitro cytotoxicity, cell cycle progression, and cellular apoptosis was determined in the MCF-7 cell line, and the NPs demonstrated a superior anti-proliferative activity associated with PTX-induced cell cycle arrest in G2/M phase and apoptosis compared to their nonresponsive counterparts. Moreover, the redox-responsive NPs were more efficacious than both free PTX and the non-redox-responsive formulation at equivalent doses of PTX in a breast cancer xenograft mouse model. This redox-responsive PTX drug delivery system is promising and can be explored for use in effective intracellular drug delivery. KEYWORDS: redox-response, paclitaxel, polymer−drug conjugates/prodrugs, polymeric nanoparticles, programmed drug release coupling macromolecular-like polysaccharides,4 peptides,5 and polymers6 to PTX. As one of the most well-known synthetic polymers, poly(ethylene glycol) (PEG), a hydrophilic polymer approved by the FDA with little toxicity and immunogenicity,7 exhibits unique features and has been used for modification of antitumor drugs for years.8 PEGylation can improve drug solubility, create a so-called “steric stabilization” effect, and provide relatively long plasma residence times.9 Moreover, it is generally accepted that PEG imparts amphiphilicity to the resultant conjugates, leading to the formation of a core−shell structure. Some researchers have focused on the development of self-assembling micelles of amphiphilic PTX prodrugs to improve the circulation behavior of native PTX.10−12 However,

1. INTRODUCTION Paclitaxel (PTX) is a potent chemotherapeutic agent isolated from the bark of the Pacific Yew tree, Taxus brevifolia, that shows promising antitumor activity against various cancers, including lung, ovarian, and breast cancer.1 Limited by its aqueous insolubility, the main clinical formulation of PTX, Taxol, contains a 1:1 ratio of Cremophor EL and ethanol. Unfortunately, Cremophor EL has been reported to be associated with various side effects, including hypersensitivity, neurotoxicity, and neuropathy.2 One strategy to potentially increase the clinical utility of PTX is to design water-soluble derivatives. The emerging polymer− drug conjugates/prodrugs have provided the possibility of overcoming some of the shortcomings of chemotherapy because the linkers between the polymers and drugs are stable in the circulatory system of the body and because the drugs can be released from the polymers into the tumor tissues/cells.3 Water-soluble polymer−PTX prodrugs have been created by © 2014 American Chemical Society

Received: Revised: Accepted: Published: 3656

June 3, 2014 September 3, 2014 September 10, 2014 September 10, 2014 dx.doi.org/10.1021/mp500399j | Mol. Pharmaceutics 2014, 11, 3656−3670

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Therefore, we prepared a PTX-loaded NP using this prodrug and free PTX via their self-assembly in an aqueous environment. In this way, PTX is located in the hydrophobic PTX inner core protected by the hydrophilic PEG outer shell. As shown in Figure 1, the designed PEG-SS-PTX/PTX NPs are

these prodrugs possess several inherent drawbacks, such as low drug loading and incomplete drug release. In addition, it has been shown that the stable PEG corona is not always beneficial for drug delivery. The coating may hinder drug release and can therefore be an obstacle in the realization of a therapeutic response.13 Intrinsic stimuli can be used to ensure successful intracellular drug release in a spatially controlled manner.14 Abnormalities in tumor cells, such as acidic pH,15−17 the presence of specific enzymes,18 and redox potential,19−21 can be used to control the behavior of nanocarriers, achieving enhanced antitumor effects. The intracellular glutathione (GSH) concentration is known to be substantially higher than that in the extracellular environment (∼10 mM vs ∼2 μM).22 Furthermore, tumor tissues are highly reducing compared to normal tissues, with a several times higher concentration of GSH in the tumor cytosol than in that of normal cells.23 On the basis of this unique intracellular redox potential, the design of reduction-responsive nanocarriers has been investigated extensively.19,24,25 Disulfide bonds, which are stable in the mildly oxidizing extracellular milieu, show rapid cleavage (over a time scale from minutes to hours) through thiol−disulfide exchange reactions with intracellular reducing molecules, especially GSH,21 and are increasingly being examined as responsive linkers for drug delivery systems.26 In particular, PTX has been conjugated to macromolecules such as PEG27 or hyaluronic acid28 through disulfide bonds and selfassembled into a core−shell structure to develop shelldetachable micelles. Drug release rate is of great importance to drug efficacy. For maximum tumor cell killing, an adequate drug concentration is required to be present over a prolonged time period.29 Furthermore, the vascular endothelial cells that support the tumor may continue to grow during rest periods from the drug, resulting in more aggressive cancers that are resistant to cytotoxic drugs;30 thus, a high concentration of drug at the tumor site for a longer period may be more effective in cancer therapy. In taking this into account, multidrug delivery in a sequential manner was explored in chemotherapy. In our previous study, drugs with different mechanisms of action were combined into one drug carrier, either through chemical conjugation or physical encapsulation. By taking into consideration the differences in their loading modes and release kinetics, a sequential killing effect of drugs could be obtained, leading to more efficient antitumor actions.31,32 However, combining drugs into one carrier presents challenges for encapsulation and for the release mode, especially for drugs with different physicochemical characteristics. Additionally, it is difficult to ensure the delivery of both drugs to the same location with a desired, relevant release pattern.29 Here, it was proposed that an increased therapeutic effect could be achieved by taking advantage of the rapid release of an entrapped drug along with the simultaneous sustained release of its conjugated form carried in a single formulation. The fast release of the initial free drug was employed to attack to tumor cells intensely, and the later released conjugated drug was employed to continue the treatment over a longer period, which was defined as programmed drug release. In this study, a polymer−drug conjugate, poly(ethylene glycol)-disulfide-paclitaxel (PEG-SS-PTX), which contains a disulfide linkage between PEG and PTX, was developed. We expected that the redox-responsive, amphiphilic conjugate would not only serve as a stimuli-responsive water-soluble prodrug for PTX but also could be used as a drug carrier.

Figure 1. Schematic representation of the self-assembly, accumulation at the tumor site, uptake by tumor cells, and triggered intracellular drug release of redox-responsive PEG-SS-PTX/PTX NPs. The programmed drug release undergoes two phases: release of loaded PTX and conjugated PTX.

expected to target tumor tissues through the enhanced permeability and retention (EPR) effect and are then taken into the tumor cells with little drug leakage because the disulfide bonds are sufficiently stable against GSH in plasma. However, upon reaching the reductive cytosol, the PEG shell rapidly detaches, achieving improved NP disassembly and intracellular payload release. The release process is expected to undergo two stages. First, the free PTX is released immediately along with disassembly of the NPs, achieving a high drug concentration in the tumor cells. Then, the previously hidden disulfide bonds become completely exposed to the reductive environment and are fully cleaved. As a result, the prodrugs will be activated and expel the conjugated PTX, thereby improving the total concentration of intracellular drug and prolonging the action time, resulting in increased antitumor efficacy. Thus, the delivery system combines several complementary functions, including (1) water-soluble prodrugs, (2) high drug content, (3) stealth character and the EPR effect, (4) redox-triggered PEG detachment, and (5) programmed drug release. 3657

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2.2.2. Synthesis of PEG-SS-MPA. Py-SS-MPA (214 mg, 1 mmol) was dissolved in CH2Cl2 followed by addition of acetic acid to adjust the pH to 2.0. Under a nitrogen atmosphere, PEG-SH (500 mg, 0.1 mmol) in CH2Cl2 was added dropwise. The reaction mixture was stirred overnight at room temperature. The solvent was evaporated under vacuum. The resulting product mixture was dissolved in water and kept in an ice bath for 3 h. The solution was filtered, and the filtrate was dialyzed (MWCO 3500) exhaustively against distilled water to remove excess Py-SS-MPA. Finally, the solution was lyophilized to obtain product and stored at −20 °C for further use. The product was characterized by Fourier transform infrared (FTIR) spectrometry. The amount of unreacted thiol groups was quantified by Ellman’s method using a spectrophotometer.34 Disulfide contents were evaluated after reduction with NaBH4 and determined by Ellman’s reagent.35 2.2.3. Synthesis of PEG-SS-PTX. PEG-SS-MPA (550 mg, 0.11 mmol) was dissolved in anhydrous CH2Cl2 and cooled to 0 °C in an ice bath. EDC·HCl (38.5 mg, 0.20 mmol) was added dropwise, and the mixture was left stirring for 20 min. PTX (8.5 mg, 0.10 mmol), DIPEA (35 μL, 0.20 mmol), and DMAP (1.2 mg, 0.01 mmol) were added, and the solution was brought back to room temperature, and the reaction was continued for 48 h. Then, the solvent was removed under reduced pressure, and the residue was dissolved in DMSO. The resulting solution was dialyzed exhaustively against ethyl alcohol followed by distilled water. After being freeze-dried, the desired product was afforded and stored at −20 °C for further use. The product was characterized by MALDI-TOF-MS and 1H NMR. The content of PTX conjugated to PEG was estimated by UV measurements based on a standard curve generated with known concentrations of PTX in methanol (λ = 228 nm).36 PEG-PTX was obtained by the same method for use as a control, with PEG-PA being replaced by PEG-SS-MPA. 2.3. Preparation of PTX Loaded PEG-SS-PTX NPs. PTXloaded PEG-SS-PTX NPs (PEG-SS-PTX/PTX) were prepared using a thin-film hydration method reported previously with slight modification.37 Briefly, 10 mg of PEG-SS-PTX and 2 mg of PTX were first dissolved in 6 mL of acetonitrile/CH2Cl2 (v/ v, 1:1). The organic solvent was removed at 37 °C by rotary evaporation for 40 min to form a thin film. PBS (2 mL) was added to rehydrate the polymer film, followed by 10 min of sonication to obtain a clear NP solution. Finally, the solution was filtered through a 0.22 μm membrane to remove precipitates. PTX-loaded PEG-PTX NPs (PEG-PTX/PTX) were also prepared for use as a control by the same method, with replacement of PEG-SS-PTX by PEG-PTX. The entrapment efficiency (EE) and drug loading (DL) were calculated as follows: EE (%) = (mass of PTX in NPs/mass of the feeding PTX) × 100; DL (%) = [mass of PTX in NP/(mass of the feeding conjugate + mass of PTX in NP)] × 100. PTX concentrations were measured by HPLC-UV at 227 nm. 2.4. Physicochemical Evaluation of PEG-SS-PTX/PTX NPs. 2.4.1. Particle Size and Morphology Analysis. The particle size and size distributions of the NPs were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS (Malvern, UK) at 25 °C. Analysis of each nanoconjugate solution was performed in triplicate. The shape and morphology of the NPs were observed with a transmission electron microscope (TEM, JEM-1230, JEOL, JAPAN). Samples were prepared by depositing NP solutions on a copper TEM grid with a carbon film and air drying at room temperature.

Here, a novel redox-responsive PEG-SS-PTX conjugate/ prodrug was synthesized, and its assembly behavior with free PTX was investigated. Subsequently, the redox-response of the NPs was studied by examining changes in their size distribution and in vitro drug-release profiles in various reducing environments. The cytotoxicity and its effect on the cell cycle and apoptosis were further characterized in the MCF-7 cell line. Finally, the therapeutic efficacy of the NPs was evaluated in tumor-bearing mice.

2. MATERIALS AND METHODS 2.1. Materials, Cell Culture, and Animals. 2,2′Dithiodipyridine (Py-SS-Py), N-(3-(dimethylamino)propyl)N′-ethylcarbodiimide hydrochloride (EDC·HCl), N,N-diisopropylethylamine (DIPEA), and 4-dimethylaminopyridine (DMAP) were purchased from J&K Scientific Ltd. (Beijing, China). 3-Mercaptopropionic acid (MPA) was purchased from ACROS Organics (New Jersey, USA). Monomethoxy poly(ethylene glycol) thiol (PEG5k-SH) and monomethoxy poly(ethylene glycol) propionic acid (PEG5k-PA) were obtained from SINOPEG Biotech Inc. (Xiamen, China). Dithiothreitol (DTT) was obtained from Lan-Yi Co., Ltd. (Beijing, China). Sulforhodamine B (SRB), trichloroacetic acid (TCA), Tris base, and Hoechst 33342 were from Sigma-Aldrich (St. Louis, MO, USA). Paclitaxel (PTX) was obtained from Haikou Pharmaceutical Co., Ltd. (Hainan, China). Cremophor EL was from BASF Corporation of Germany (from a local agent in Shanghai, China). The FITC-labeled annexin V/propidium iodide apoptosis detection kit and in situ apoptosis detection kit (FITC-labeled POD) were purchased from KeyGEN Biotech Co., Ltd.(Nanjing, China). All other reagents and solvents were analytical or HPLC grade. The human breast cancer cell line MCF-7 was acquired from the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS, Beijing, China). Cells were grown in RPMI-1640 (Macgene Biotech Co., Ltd., Beijing, China) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin, 100 unit/mL, plus streptomycin) at 37 °C in a 5% CO2 atmosphere. Female BALB/c nude mice (6−8 weeks old, 18−20g) were purchased from Vital River Laboratory Animal Center (Beijing, China) and kept under SPF conditions in the Laboratory Animal Unit of the Peking University Health Science Center with free access to standard food and water. All of the animal experiments adhered to the principles of the Institutional Animal Care and Use Committee of Peking University. 2.2. Synthesis of PEG-SS-PTX. 2.2.1. Synthesis of 2Pyridyl-2-carboxyethyl Disulfide (Py-SS-MPA). Py-SSMPA was synthesized and purified by modifying a literature method.33 To a solution of Py-SS-Py (3.75 g, 17 mmol) in 30 mL of ethanol containing 0.4 mL of acetic acid was added MPA (0.9 g, 8.5 mmol) in 20 mL of ethanol dropwise with vigorous stirring. After 2−3 h, the solution was evaporated under reduced pressure to yield a viscous yellow oil. This crude product was purified by a basic Al2O3 column. The column was washed with a 3:2 mixture of CH2Cl2/ethanol to elute starting materials and byproducts and was then eluted with solvent containing 4% acetic acid to obtain the desired product. The solvent was removed under high vacuum. The resulting viscous oil was dissolved in warm water and placed under 0 °C for 3 h to obtain a precipitate. The solid residue was collected by filtration and dried in vacuo overnight. This product was characterized by 1H NMR and ESI/MS. 3658

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2.4.2. X-ray Diffraction Analysis. X-ray diffraction (XRD) patterns of the pure PTX, conjugates, physical mixtures, and PTX-loaded NPs were determined using an X-ray diffractometer (XRD; RAPID-S, Rigaku Denki Co., Ltd., Japan). The investigation used Ni-filtered Cu Kα radiation with a 4 °C/min scanning rate at 25 °C. 2.4.3. Differential Scanning Calorimetry Analysis. Differential scanning calorimetry analysis (DSC) of pure drug, conjugates, physical mixture of PTX and PEG-SS-PTX, and PTX-loaded NPs was conducted using a DSC Q2000 (TA Instruments, New Castle, DE, USA) under a nitrogen atmosphere. Approximately 10 mg of sample placed in sealed aluminum pans was scanned at a rate of 10 °C/min from 25 to 225 °C. The different thermal transitions were analyzed. 2.4.4. 1H NMR. To evaluate the dispersion state of PTX in the NPs, the NP solution was freeze-dried and reconstituted in deuterated water (D2O) or deuterated DMSO (DMSO-d6), and 1H NMR was recorded on a Bruker AVANCE III400 (400 MHz) instrument at room temperature. 2.4.5. Physical Stability Studies of PEG-SS-PTX/PTX NPs. The physical stability of the PEG-SS-PTX/PTX NPs and PEG-PTX/PTX NPs was investigated by DLS. The samples were stored at 4 or 37 °C. At predetermined time points, samples were subjected to particle size analysis as described above. 2.4.6. Drug-Release Studies of PEG-SS-PTX/PTX NPs. The drug-release behaviors of the PEG-SS-PTX/PTX NPs and PEG-PTX/PTX NPs were investigated by a dialysis method. The NP solutions were sealed in preswollen dialysis bags (MWCO = 14 000 Da) and immersed in PBS (pH 7.4, 0.1 M) with 1.0 M sodium salicylate38 at 37 °C (with a final free PTX concentration of 0.3 mg/mL), followed by shaking at a speed of 100 rpm. At designated time intervals, samples (0.5 mL) were withdrawn and replaced with the same volume of fresh release medium. The PTX content in the release buffer was measured by high-performance liquid chromatography (HPLC, Shimadzu LC-10AT system, Japan) equipped with a Phenomenex C18 column (5 μm particle size, 250 mm × 4.6 mm). The mobile phase consisted of acetonitrile and water (50:50, v/v) with a flow rate of 1.0 mL/min. The detection wavelength was 227 nm, and the sample injection volume was 20 μL. Each experiment was carried out in triplicate. 2.5. Redox-Responsiveness Assay of PEG-SS-PTX/PTX NPs. 2.5.1. Reduction Triggered Destabilization of PEGSS-PTX/PTX NPs. The destabilization of redox-sensitive PEGSS-PTX/PTX NPs in response to DTT in PBS (pH 7.4, 0.1 M) was monitored by DLS measurement with PEG-PTX/PTX NPs as a control. Briefly, a 2 mL suspension of PEG-SS-PTX/ PTX NPs or PEG-PTX/PTX NPs was kept in a glass cell with or without 10 mM DTT. The samples were placed in a shaking bed at 37 °C with a rotation speed of 100 rpm. At given intervals, the change in size of the NPs was traced by DLS. 2.5.2. Reduction Triggered Drug Release of PEG-SSPTX/PTX NPs. The in vitro reduction-triggered drug-release characteristics of PEG-SS-PTX/PTX NPs were studied using a dialysis method (as described in Section 2.4.6) in two different release media, i.e., media with or without 50 mM DTT. Each experiment was carried out in triplicate. 2.6. In Vitro Cell Experiments for PEG-SS-PTX/PTX NPs. 2.6.1. In Vitro Cytotoxicity. The in vitro cytotoxicity of the NPs was evaluated using MCF-7 cells with the SRB assay.39 In brief, approximately 5000−7000 cells/well were seeded in 96-well plates and incubated at 37 °C in a 5% CO2 atmosphere

for 24 h. Then, the medium was removed, and free PTX or different NPs of serial PTX concentrations was added (the concentrations were calculated as free PTX). After 48 or 72 h, cells were fixed by introducing 200 μL of 10% TCA (w/v) at 4 °C for 1 h, and the plates were then washed with water and air dried. Subsequently, 100 μL of SRB was added, and staining took place at room temperature for 30 min, followed by rinsing with 1% acetic acid (v/v) and drying. Finally, 200 μL of Tris base (10 mM) was added, and the plates were shaken for 30 min. The absorbance was measured using a 96-well plate reader (BioRad, 680) at 540 nm. The IC50 values were calculated from the dose−effect curves. To estimate the cytotoxicity mediated by GSH, the intracellular level of GSH was manipulated by pretreatment with glutathione ethyl ester (GSH-OEt).22 MCF-7 cells (7000−10 000 cells/well seeded in a 96-well plate) were preincubated for 2 h with cell culture media containing 10 mM GSH-OEt. Cells without GSH-OEt pretreatment were used as a control. Subsequently, the cells were washed by PBS three times. Different NP solutions (at a PTX concentration of 25 ng/mL) were then added. After incubation for 24 and 48 h, the cytotoxicity of the NPs was evaluated by a SRB assay as described above. The in vitro cytotoxicity of different conjugates as well as the cytotoxicity mediated by GSH was also evaluated using MCF-7 cells by the same method, except that the concentration of conjugates in the GSH-associated cytotoxicity assay was 50 ng/ mL. 2.6.2. Cell Cycle Analysis. The cell cycle was analyzed by propidium iodide (PI) DNA staining.39 Briefly, MCF-7 cells seeded on a 6-well plate were treated with PTX, PEG-SS-PTX/ PTX NPs, and PEG-PTX/PTX NPs (free PTX concentration of 20 nM) at 37 °C for 12 h. Cells incubated with RPMI-1640 served as a control. At the end of the treatment, cells were collected by centrifugation, washed twice with cold PBS, and fixed with 70% alcohol overnight at 4 °C. Cells were washed to eliminate alcohol, incubated with DNase-free RNase A (100 mg/mL) for 30 min at 37 °C, and then stained with PI solution (50 mg/mL) for 30 min in the dark. Cell cycle profiles were studied using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). The data were analyzed through FCS Express V 3 software. Each assay was carried out in triplicate. 2.6.3. Cell Apoptosis Analysis. For the analysis of apoptosis, MCF-7 cells with different fomulations (free PTX concentration of 8 nM) were incubated for 48 h. After incubation, cells were harvested and washed twice with cold PBS. Then, 1 × 105 cells were collected by centrifugation and resuspended in 200 μL of binding buffer. Annexin V-FITC and PI (200 μg/mL) were added, and the cells were incubated in the dark for 15 min at 37 °C. The stained cells were analyzed using a flow cytometer (Becton Dickinson, San Jose, CA, USA). Data analysis was performed using Cell-Quest software (Becton Dickinson). 2.7. In Vivo Antitumor Efficacy and Toxicity Evaluations for PEG-SS-PTX/PTX NPs. Female BALB/c nude (nu/ nu) mice (6−8 weeks, 18−22 g) were inoculated subcutaneously in the right armpit with MCF-7 cells (5 × 106). Mice were randomly assigned to five groups treated with saline (control), Taxol (free PTX at dose 10 mg/kg), Taxol (free PTX at dose 7 mg/kg), PEG-PTX/PTX NPs (free PTX at dose 7 mg/kg), and PEG-SS-PTX/PTX NPs (free PTX at dose 7 mg/kg). Administration via tail vein injection was carried out every other day for a total of 4 doses, which was initiated when 3659

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tumor exceeded 50 mm3. Tumor width, tumor length, and body weight were also recorded every day. The tumor volumes were determined using the equation (width2 × length)/2.40 At the end point, mice were sacrificed, and tumors were harvested and imaged. The in vivo antitumor efficacy of the PTX conjugates was evaluated by the same method, except that the mice were randomly divided into four groups treated with saline, Taxol, PEG-SS-PTX, and PEG-PTX, all administered at the same PTX dose of 10 mg/kg. Hematoxylin and eosin (H&E) staining of heart, liver, spleen, lung, and kidney sections was conducted for the histological study of the toxicity of the different treatments. Tumor sections (4 μm in thickness) were stained by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling (TUNEL), using the protocol from the in situ apoptosis detection kit. Briefly, the tumor sections were deparaffinized and dehydrated and then incubated with a proteinase K working solution at 37 °C for 30 min. The slides were washed with PBS (pH 7.4) twice. The positive control was incubated with DNase I at 37 °C for 10 min. The slides were incubated with TdT solution and Streptavidin-FITC labeling solution at 37 °C for 60 and 30 min, respectively (the negative control was treated without TdT). The samples were washed with PBS (pH 7.4) three times and incubated with Hoechst 33342 for 30 min at 37 °C, followed by washing with PBS (pH 7.4) three times. The slides were mounted and analyzed microscopically (Leica, Heidelberg, Germany) 2.8. Statistical Analysis. Data are shown as the mean ± standard deviation (SD). Statistical evaluation was performed by two-tailed Student’s t test and one-way analysis of variance (ANOVA). p < 0.05 was considered to be statistically significant, and p < 0.01 was considered to be highly significant.

Figure 2. Synthetic routes, reagents, and conditions. Synthesis of PySS-MPA (A), PEG-SS-MPA (B), and PEG-SS-PTX (C). (i) Ethanol/ AcOH, rt, 2−3 h; (ii) CH2Cl2, pH 2.0, rt, 12 h; (iii) EDC·HCl, DIEPA, DMAP, CH2Cl2, rt, 48 h.

3. RESULTS AND DISCUSSION In our previous study, PEG-PTX was synthesized. Due to its amphiphilic nature, PEG-PTX can self-assemble in aqueous solution and form nanomicelles (∼10 nm) or aggregate into large particles (>600 nm); neither of these possibilites are suitable for drug delivery. However, when PTX was loaded with PEG-PTX, NPs with sizes ranging from 100 to 200 nm were obtained, which revealed that adding hydrophobic PTX to PEG-PTX conjugates improved the assembly of NPs.41 However, the PEG-PTX conjugates showed little anticancer activity and could serve only as a carrier. To preserve the activity of PTX, the theory of stimuli-responsive drug release was adopted, and a disulfide linkage was introduced to release the active PTX on demand. The newly developed PEG-SS-PTX could be used as both a prodrug and a drug carrier. After assembly with free PTX, a NP system with a high drug content, low risk of drug leakage, and redox-triggered shell detachment can be obtained. Moreover, due to the dissimilar release rates between the entrapped PTX and the conjugated PTX, a programmed drug-release profile can be achieved, thereby prolonging the drug’s active time, increasing the overall intracellular drug concentration, and, as a result, enhancing the antitumor efficacy. 3.1. Synthesis and Characterization of PEG-SS-PTX. The redox-responsive PEG-SS-PTX copolymer was synthesized in three steps, as shown in Figure 2. Py-SS-MPA was first prepared via a thiol−disulfide exchange reaction. The reaction was performed in an ethanol/acetic acid system, with the simultaneous dropping of MPA into the Py-SS-Py solution. The product yield was 30.6%. 1H NMR (400 MHz, DMSO-d6,

Figure 3A) indicated the structure of Py-SS-MPA, and the properties were in accordance with the results reported in ref 33: δ, 2.59 (2H, t, J = 7.0), 2.98 (2H, t, J = 7.0), 7.23 (1H, ddd, J = 7.1, 4.8, 1.2), 7.75 (1H, d, J = 8.0), 7.8 (1H, td, J = 7.9, 1.4), 8.4 (1H, d, J = 4.9). The ESI-MS spectrum exhibited a peak at m/z 215.9 for C8H9NO2S2 (data not shown), which was consistent with the calculated value. Subsequently, the obtained Py-SS-MPA was reacted with PEG-SH by the thiol−disulfide exchange reaction, resulting in the formation of PEG-SS-MPA. To ensure complete reaction, excess Py-SS-MPA was used (10:1 molar ratio of Py-SS-MPA/ PEG-SH). The resulting copolymer was purified by repeated washing and subsequent dialysis against water. The NMR spectrum of the product indicates the successful conjugation of PEG-SH to Py-SS-MPA (Figure 3B), in which new peaks belonging to methylene in Py-SS-MPA at 3.0 ppm were observed. In the FTIR spectrum of PEG-SS-MPA (Figure S1), a new peak appeared at 1770 cm−1 and a broad peak appeared at 3431 cm−1, indicating the presence of carboxyl groups. In addition, the characteristic C−O−C stretching vibrations of the PEG side chains near 1110 cm−1 also remained, confirming the structure of the obtained PEG-SS-MPA. Ellman’s assay quantitatively showed that the product possesses a high disulfide functionality of 196 μmol/g and a low thiol group content of 3 μmol/g, which illustrated that 98.1% of PEG-SH successfully transformed into PEG-SS-MPA. In addition, the pyridyl peak of the Py-SS-MPA reactant was not observed in the UV spectrum of the product (Figure S2A), further proving the completion of the reaction. 3660

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Figure 3. 1H NMR spectra of Py-SS-MPA (A), PEG-SH and PEG-SS-MPA (B), and PEG-SS-PTX (C); MALDI-TOF spectra of PEG-SS-MPA and PEG-SS-PTX (D). In panels B and C, * indicates methylene group protons in Py-SS-MPA and PTX, respectively.

In this study, a PEG-PTX conjugate with a similar composition except for a disulfide linkage was also prepared and used for comparison. 3.2. Fabrication and Characterization of PEG-SS-PTX/ PTX NPs. 3.2.1. Particle Size and Morphology Analysis. PEG-SS-PTX/PTX NPs were prepared by a modified thin-film hydration method.37 DLS and TEM were used to confirm the formation of PEG-SS-PTX/PTX NPs. As shown in Figure 4A,B and Table 1, the mean diameters of the PEG-SS-PTX/PTX NPs and the PEG-PTX/PTX NPs in water were approximately 108 and 130 nm, respectively, and the drug-loading efficiencies for both NPs were higher than 15%, without significant differences. Recently, many amphiphilic copolymers have been synthesized for the delivery of PTX. Unfortunately, the majority of them have exhibited low drug-loading capacities. A successful nanodelivery system should have a high drug-loading capacity, thereby reducing the quantity of matrix materials for administration.45 Jiang et al. reported novel PTX-loaded NPs with high DL and EE values of 6.4 and 93%.46 It was concluded that with an increase in the amount of PTX, the DL increases, whereas the EE decreases.47 In this study, the thin-film method was used to prepare PTX-loaded NPs, and both a successful DL and high EE were achieved. TEM micrographs revealed that the NPs had a spherical morphology with an average size of 70 nm, which was smaller than that obtained from DLS. The larger diameter determined by the DLS measurement may be due to the dehydration of the

Next, PTX was directly conjugated to PEG-SS-MPA, using EDC/DMAP as a coupling agent, to form PEG-SS-PTX. A 75.8% yield was obtained. Figure 3C shows the 1H NMR spectra of PEG-SS-PTX in DMSO-d6. The characteristic peaks of PTX were clearly displayed, with aromatic (7.0−8.0 ppm), acetyl (2.1−2.5 ppm), and methyl (1.0−1.2 ppm) protons. The structure of PEG-SS-PTX was also verified by MALDI-TOF MS analysis. The MW of PEG-SH is 4900. After conjugation with PTX, the MW shifted to 5700−5900 (Figure 3D), which was in agreement with the theoretical MW of PEG-SS-PTX. Furthermore, the full-scan UV result from 190 to 400 nm indicated that PEG-SS-PTX had a maximal absorbance at 227 nm, which was attributed to the conjugated PTX (Figure S2A). Additionally, the reaction was shown to be complete after 48 h, as demonstrated by HPLC, in which no PTX peak was found (Figure S2B). According to previous reports, it appears that C2′−OH in PTX preferentially reacted, rather than C7′−OH, because it is less sterically hindered and that C7′−OH of PTX can be derivatized only under strong conditions.36,42 Esters at C2′−OH have proved to be more labile and can be synthesized selectively by fixing the molar ratio of carboxyl reagents and the EDC agent without protecting C7′−OH.43,44 Therefore, we speculate that PTX derivatives at C2′−OH were prepared selectively under the reaction conditions used here. The PTX content of PEG-SS-PTX was found to be 10.6% by an established ultraviolet−visible method. All of these results confirmed the well-defined structure of PEG-SS-PTX. 3661

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Figure 4. Characteristics of PEG-SS-PTX/PTX NPs. Particle size and morphology determined by DLS analysis and TEM images based on PEG-SSPTX/PTX NPs (A) and PEG-PTX/PTX NPs (B). Scale bars = 100 nm. (C) XRD patterns of pure PTX (a), PEG-SS-PTX conjugates (b), physical mixture (c), and PEG-SS-PTX/PTX NPs (d). 1H NMR spectra of PTX in DMSO-d6 (D), PTX/PEG-SS-PTX NPs in D2O (E), and PEG-SS-PTX/ PTX NPs in DMSO-d6 (F). * indicates free PTX protons.

Table 1. Characteristics of PEG-SS-PTX/PTX NPs and PEG-PTX/PTX NPsa nanoparticles

sizesb (nm)

PDIc

DLd (wt %)

EEd (%)

PEG-SS-PTX/PTX NPs PEG-PTX/PTX NPs

108 ± 8.07 133 ± 3.75

0.26 ± 0.04 0.21 ± 0.05

15.7 ± 0.10 15.2 ± 0.15

93.3 ± 0.74 89.9 ± 1.04

Data are represented as mean ± SD (n = 3). bMean diameters of NPs determined by DLS. cPolydispersity index of micelles size. dDL and EE are abbreviations for drug-loading and entrapment efficiency, respectively.

a

crystalline peaks in the NPs indicated that the PTX was present in an amorphous state due to the process of encapsulation. The 1H NMR spectra of free PTX in DMSOd6 and PEG-SS-PTX/PTX NPs in DMSO-d6 and D2O were also investigated. A typical 1H NMR spectrum of free PTX in DMSO-d6 is shown in Figure 4D. When PTX was encapsulated into the NPs, the resonance peaks corresponding to PTX at 1.2 and 1.0 ppm and the aromatic signals at 7.0 to 8.0 ppm were hardly seen in D2O because of its inefficient mobility in D2O (Figure 4E). However, in DMSO-d6, the characteristic PTX

NPs and the shrinkage of the PEG shell induced by water evaporation under the high vacuum conditions used before TEM observation.48 3.2.2. State Analysis of PTX Encapsulated in NPs. To evaluate the PTX state in the NPs, XRD, DSC, and 1H NMR analyses were performed. XRD and DSC are used to elucidate whether the drug is in a crystalline or amorphous state. Figures 4C and S3 illustrate the XRD spectra and DSC thermograms of pure PTX (a), PEG-SS-PTX conjugates (b), physical mixtures (c), and PEG-SS-PTX/PTX NPs (d). The absence of 3662

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Figure 5. Comparison of stability of the PEG-SS-PTX/PTX NPs (A) and PEG-PTX/PTX NPs (B) as a function of the particle size measured by DLS at 4 °C over a period of 4 weeks. (C) In vitro release profiles of PTX from the two NPs in PBS (pH 7.4) for 24 h (n = 3). Size changes of PEGSS-PTX/PTX NPs (D) and PEG-PTX/PTX NPs (E) in response to DTT in PBS, determined by DLS. (F) Redox-triggered release of PTX from the PEG-SS-PTX/PTX NPs in PBS (pH 7.4) (n = 3).

For long-term storage (4 weeks) at 4 °C, there was no notable aggregation in the PTX-loaded NP solution, and the mean diameter of the NPs did not change remarkably (Figure 5A,B), indicating the thermodynamic stability of PTXencapsulated NPs under aqueous conditions. In contrast, when nanoprecipitation was performed using the free drug instead of the conjugate, PTX precipitated in the aqueous phase immediately and formed needle-like crystals.52 It has been proved that PEG can decrease the amount of attraction between NPs by increasing the steric distance between them and increasing the hydrophilicity via ether repeats, forming hydrogen bonds with the solvent.9 Additionally, PEG decreases the tendency of NPs to aggregate by minimizing the surface energy of NPs and the van der Waals attraction.9 Therefore, an appropriate density of PEG and PEG length on the NP surface may enhance the stability of the NPs in an aqueous environment. It should be noted that PEG-SS-PTX/PTX NPs exhibited a slightly higher stability than PEG-PTX/PTX NPs with no size change during a period of 4 weeks, which could be

peaks were clearly observed (Figure 4F). This phenomenon might be due to the formation of a core−shell structure in water in which the hydrophobic PTX is trapped in the core and isolated by the hydrophilic PEG shell,49 whereas the conjugate would be fully dissolved in DMSO. These results are consistent with the 1H NMR studies of other PTX polymer NPs reported.49,50 All of these observations indicated that PTX was successfully encapsulated into the hydrophobic inner core of the polymeric NPs. 3.2.3. Physical Stability Studies of PEG-SS-PTX/PTX NPs. The physical stability of NPs was investigated by recording the particle size changes by DLS. After incubation with PBS at 37 °C for 6 h, there were no significant changes in the average particle size (data not shown), which ensured that the NPs would be stable during use. In the clinic, nanomedicines are usually infused for a relatively long time, for example, Genexol-PM is intravenously infused over 3 h for each dose.51 Therefore, the stability of the system during infusion is very important. 3663

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Figure 6. In vitro cytotoxicity toward the MCF-7 cell line after incubation with various concentrations of free PTX, PEG-SS-PTX/PTX NPs, and PEG-PTX/PTX NPs alone for 48 h (A) and 72 h (B). GSH-OEt-mediated cytotoxicity against MCF-7 cells after incubation with PEG-SS-PTX and PEG-PTX conjugates (C) or with PEG-SS-PTX/PTX NPs and PEG-PTX/PTX NPs (D) for 24 and 48 h. Data represent the mean ± SD (n = 6).

related to the molecular structures of the prodrugs: PEG-SSPTX is a conjugate of PTX coupling with PEG through the disulfide group and thus the sulfur might provide electron pairs, interacting with the benzene of PTX and forming a relatively stable NP system. 3.2.4. Drug-Release Study of PEG-SS-PTX/PTX NPs. The drug-release behavior was also evaluated in terms of the drug-release profile. According to Figure 5C, drug release from the PEG-SS-PTX/PTX NPs reached 66.2% at approximately 24 h, whereas the same level of drug release from the PEG-PTX/ PTX NPs required only 2 h. Moreover, the PTX released from both the PEG-SS-PTX and the PEG-PTX conjugates was lower than 4% at 72 h (Figure S4 A), which was negligible in comparison with the free PTX released from the drug-loaded NPs. The results indicated that the PEG-SS-PTX/PTX NPs exhibited a much slower release rate than that of the PEGPTX/PTX NPs, which was consistent with the above sizechange investigation. This may result from the stronger interaction between PTX and PEG-SS-PTX, as discussed above. It has been reported that the fast release of insoluble drugs from carriers may induce subsequent drug precipitation or their transfer to plasma proteins, which decreases their biological reactions.53 The slower release of PTX from the PEG-SS-PTX/PTX NPs might prevent its premature release into the blood, which would be beneficial for in vivo treatments. 3.3. In Vitro Redox-Responsiveness Evaluations of PEG-SS-PTX/PTX NPs. 3.3.1. Particle Size Changes of NPs Triggered by Reducing Conditions. The disulfide bridge linkage between the hydrophobic PTX inner core and the hydrophilic PEG outer shell makes the PEG-SS-PTX/PTX NPs prone to disassembly by reducing agents. To prove the redox

response, the size changes of the NPs treated with 10 mM DTT were recorded by DLS at various time intervals. As shown in Figure 5D, when treated with DTT, fast aggregation was observed for PEG-SS-PTX/PTX NPs, in which the size increased from 120 to 819 nm in 2 h. In contrast, the NP solution without DTT treatment showed no significant size changes. The data were similar to results published previously.48 Such variation in the size is probably due to the reductive cleavage of disulfide linkages, which results in the quick disassembly of the NPs as well as activation of a few prodrug molecules, followed by an enhancement in the hydrophobic interaction of the PTX inner core. On the other hand, the PEG-PTX/PTX NPs were stable in structure, with nearly no size variation regardless of the presence of DTT (Figure 5E). 3.3.2. PTX Release from PEG-SS-PTX/PTX NPs Triggered by Reducing Conditions. The in vitro PTX-release behavior of drug-loaded redox-responsive NPs was studied by a dialysis method. As shown in Figure 5F, without DTT treatment, the accumulative release of PTX reached 64.4% within 6 h, and the subsequent release rate was much slower. However, after adding DTT, the accumulative release of PTX dramatically accelerated, reaching 97.2% in 6 h. As discussed previously, the disassembly of the NPs in the presence of DTT is due to the cleavage of disulfide bonds. In the early period of release, the drug-release rate was very fast, which could be mainly attributed to the release of the loaded, free PTX caused by the accelerated disassembly of the NPs in the reductive environment. In addition, the conjugated PTX cleaved from PEG-SS-PTX conjugates was detectable in the presence of DTT, although the amount was very small (13.6% up to 24 h, 3664

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both in vitro and in vivo, despite the fact that the C-2′ position of PTX was occupied.54 Similarly, an octa-arginine−Taxol conjugate developed by introducing a disulfide linker at the C2′ position was proven to be effective against Taxol-resistant cell lines.55 These results proved that PEG-SS-PTX conjugates could serve as prodrugs to exert antitumor action. To test whether the cleavage of PTX from the PEG-SS-PTX conjugates is due to the high cellular redox potential, not enzymes or other factors, we introduced GSH-OEt to investigate the effect of redox response on tumor inhibition efficacy. It is well-documented that GSH-OEt can penetrate cellular membranes and rapidly reach a high intracellular concentration of GSH through ethyl ester hydrolysis in the cytoplasm.22 MCF-7 cells were preincubated with 10 mM GSH-OEt for 2 h to manipulate the intracellular concentration of GSH and were further incubated with nanoconjugate solutions containing 0.05 μg/mL PTX for 24 and 48 h. As shown in Figure 6C, with GSH-OEt preincubation, the inhibitory effect of the sensitive PEG-SS-PTX nanoconjugate improved significantly after 48 h (p < 0.01). However, GSHOEt pretreatment did not change the cytotoxicity of PEG-SSPTX after 24 h, which indicated that it required time to cleave PTX from the conjugates and that a higher intracellular GSH concentration helped to accelerate drug release. As a comparison, the inhibition effect of PEG-PTX was not affected by the GSH-OEt either after 24 or 48 h, further supporting our expectation that PEG-SS-PTX prodrug could be activated by the cellular redox potential. The GSH-dependent cytotoxicity of NPs was also investigated. As shown in Figure 6D, after GSH-OEt addition, the inhibitory effect of PEG-SS-PTX/PTX NPs improved significantly when incubated for both 24 and 48 h (p < 0.001 and p < 0.01, respectively), supporting the redox responsiveness of the PEG-SS-PTX/PTX NPs. It is worth noting that GSH-OEt significantly impacted the cytotoxicity of the sensitive NPs after only 24 h, which was much less time than that for the sensitive conjugates. This means that the inhibition effect of PEG-SS-PTX/PTX NPs in the first 24 h period was mainly attributed to the loaded, free PTX released from the NP core, whereas the conjugated PTX began to display cytotoxicity after 48 h. These results proved our expectation of programmed drug release (that the loaded, free drug and activated prodrug released sequentially), resulting in a programmed killing effect against the tumor cells. However, the inhibition effect stayed nearly unchanged after GSH-OEt preincubation, indicating that the enhanced cytotoxicity of the sensitive NPs can be achieved in the presence of a higher intracellular GSH concentration via efficiently triggering the degradation of the disulfide bridge linkage. 3.4.2. Cell Cycle Analysis. It has been reported that PTX can impair mitosis and induce cell cycle arrest.56 Therefore, increased G2/M phase arrest indicates cell division inhibition and cell growth restraint.57 The cell cycle analysis results are shown in Figure 7A. In contrast to the control group (15.4%), free PTX caused a markedly increased accumulation of G2/M phase cells (60.3%). The percentage of PEG-SS-PTX/PTX NPtreated and PEG-PTX/PTX NP-treated cells in G2/M phase was 33.9 and 19.7%, respectively. Compared with that of free PTX, the decreased activity of PEG-SS-PTX/PTX caused by PEGylation was designed to minimize the cytotoxicity of the PTX conjugate to normal cells. The antitumor efficiency can be exerted effectively by a longer treatment time. In contrast, the insensitive counterparts showed a much lower mitotic-arresting effect, almost the same as that for the control group. Therefore,

Figure S4B), which also helped to accelerate the disassembly of the NPs. In the later period, the free PTX was almost entirely released; the much slower release rate was due to the remaining large amount of conjugated PTX released from prodrugs (seen in Figure S4B), which was also indicated by the final accumulative released amount from the DTT-treated group that had a value larger than 100%. These results were in accordance with our programmed release expectation, namely, that the entrapped and conjugated PTX was released successively as a result of the NP disassembly and the subsequent prodrug activation. These results suggest that the redox-responsive NPs will minimize drug leakage into the blood and achieve rapid drug release in the reducing tumor environment. Moreover, a twophase programmed drug release can be obtained, which may prolong the active time and enhance the total quantity of the active drug, resulting in increased efficiency. Therefore, the redox-responsive NPs may be promising for use in tumortargeting drug delivery. 3.4. In Vitro Cell Experiments for PEG-SS-PTX/PTX NPs. 3.4.1. In Vitro Cytotoxicity. The in vitro cytotoxicity of the PTX free drug, PEG-SS-PTX/PTX NPs, and PEG-PTX/ PTX NPs toward MCF-7 human breast cancer cells was estimated using the SRB method. As shown in Figure 6A,B, the free PTX and the two NP formulations exhibited cytotoxicity in a time- and dose-dependent manner. As expected, the cytotoxicity of redox-sensitive NPs was significantly higher in comparison with that of insensitive ones at both incubation time points. The degradation of reducible conjugates in the reductive cytosol may explain the elevated cytotoxicity. It is worth noting that the PEG-SS-PTX/PTX NPs exhibited relatively lower toxicity (IC50 = 27.5 ng/mL) compared to that of free PTX (IC50 = 19.2 ng/mL) at 48 h, but they demonstrated higher toxicity (IC50 = 1.80 ng/mL) than free PTX (IC50 = 3.10 ng/mL) at 72 h. This is probably because free PTX can be readily transported into cells by a passivediffusion mechanism, whereas the NPs must be internalized by endocytosis, resulting in delayed intracellular drug delivery and lower cell inhibition efficacy. However, PTX release from the PEG-SS-PTX/PTX NPs is accelerated after the reductive breakage of the NPs by GSH within the cytosol. In addition, the conjugated PTX found on prodrugs can be liberated as time passes, as confirmed in the above NP-release studies. Therefore, the total amount of active PTX was increased in the end, and the cytotoxic potential of the PEG-SS-PTX/PTX NPs varied at different time points, which proved the expectation of programmed drug release. To further prove that the programmed drug release is the result of the different release rates between free, loaded PTX and PEG-SS-PTX conjugates, the cytotoxicity of PEG-PTX and PEG-SS-PTX was also monitored. As shown in Figure S5, the PTX group showed the highest cytotoxicity, indicating that modification of PTX by PEG negatively affects its cytotoxicity because PEG inhibits the cellular uptake of the conjugate.49 However, the cytotoxicity of PEG-SS-PTX was significantly higher than that of PEG-PTX, indicating that redox-responsive PEG-SS-PTX could release active drug in a reductive intracellular environment. The released SH-PTX was still cytotoxic after redox-mediated cleavage, which is consistent with the assertion of previous reports that esterification at either C-2′ or C-7′ does not significantly influence the activity of PTX.42 In 2002, the disulfide prodrugs of PTX were reported to exert equivalent or even superior activities to that of PTX 3665

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Additionally, in consideration of effective tumor inhibition, Taxol administered at a dose of 10 mg/kg, which is close to its maximum tolerated dose (MTD), was selected as another control. As shown in Figure 8A,B,D, at the same free-PTX dose (7 mg/kg), PEG-SS-PTX/PTX NPs exhibited superior tumorgrowth inhibition compared to that of the PEG-PTX/PTX NPs (p < 0.001), which can be explained by the accelerated release rate of intracellular PTX, resulting from the redox-responsive NP disassembly. Moreover, the PEG-SS-PTX conjugate could serve as a prodrug to exert further antitumor efficacy, whereas PEG-PTX showed no activity (Figure S6); therefore, a difference in tumor inhibition was obtained, which was in accord with the observations from the in vitro studies of the NPs. It should be further noted that the PEG-SS-PTX/PTX NPs exhibited a much higher antitumor potency than that of Taxol at a dose of 7 mg/kg and even showed a similar efficacy to that of Taxol at a higher dosage (10 mg/kg). These results indicated that PEG-SS-PTX/PTX NPs may be able to achieve a significant inhibitory effect with a relatively low dosage, which gives this nanomedicine a high MTD. Despite their lower in vitro cytotoxicity, PEG-SS-PTX/PTX NPs showed enhanced in vivo therapeutic efficacy, which might be due to the higher amount of PTX reaching the tumor tissue via the stealth character of these NPs and the EPR effect. In addition, once reaching the tumor site and particularly within the tumor cells, the reducing environment is expected to remove the PEG shell, accelerating the disassembly of the NPs and the subsequent prodrug activation. The triggered, programmed drug release might exert a rapid and significant antitumor effect by first releasing the entrapped PTX and then maintaining a modest tumor inhibition effect by releasing the conjugated PTX, achieving a superior therapeutic efficiency. The toxicitt of the NPs was assessed by analyzing their effect on body weight (Figure 8C). Severe body weight loss (25%) was observed after treatment with Taxol, which was likely attributed to the use of Cremophor EL and ethanol as the vehicle for PTX and the rapid high peak level of PTX in the blood.58 However, decreased body weight loss was observed in the PEG-SS-PTX/PTX NP groups (16%), demonstrating that the redox-responsive NPs had a much lower toxicity than that of Taxol. The formulation of PEG-SS-PTX/PTX contained only two components, PEG and PTX, which minimized the possible toxicity induced by other foreign bodies. Furthermore, to verify whether the PEG-SS-PTX/PTX NPs impaired tissue function and morphology, H&E stained sections of the main organs were examined. As presented in Figure 8E, in the PEG-SS-PTX/PTX NP-treated group, all major organs, including the heart, liver, spleen, lung, and kidney, were normal compared with those of the PBS control group, without obvious histopathological abnormalities, degenerations, or lesions, indicating that PEG-SS-PTX/PTX NPs were biocompatible and did not induce severe damage. This further proved that the low toxicity of PEG-SS-PTX/PTX is of great value for a high MTD. It has been demonstrated that Abraxane shows a rapid elimination of PTX from the circulation and does not improve the pharmacokinetics of PTX,59 but it increases the safety and MTD of PTX in human patients.60 When given i.v. every 3 weeks, the MTD in humans for Abraxane was 300 mg/m2, considerably higher than the 175 mg/m2 MTD for CremophorPTX.61 The redox-responsive NPs that we developed here can

Figure 7. Effect of treatment with free PTX, PEG-SS-PTX/PTX NPs, and PEG-PTX/PTX NPs on the cell cycle distribution of MCF-7 cells (12 h treatment; equivalent PTX concentration of 20 nM, n = 3) (A) and on apoptosis (48 h treatment; equivalent PTX concentration of 8 nM, n = 3), determined by flow cytometry using annexin V/propidium iodide double staining (B).

the redox-responsive treatment is efficient in promoting mitotic arrest. 3.4.3. Cell Apoptosis Analysis. To characterize the apoptotic effect of PTX and redox-responsive NPs, a quantitative apoptotic assay was performed. According to Figure 7B, the percentage of free PTX-treated cells in early and late apoptosis was 32.45 and 17.08%, respectively (Figure 7B,b). PEG-PTX/PTX NPs caused 12.75 and 7.62% of cells to be in early and late apoptosis (Figure 7B,d), respectively, whereas those of PEG-SS-PTX/PTX NPs increased to 21.58 and 10.19%, respectively (Figure 7B,c). These results were consistent with the above in vitro cytotoxicity and cell cycle analysis, and they indicated that the PEG-SS-PTX/PTX NPs substantially increase the extent of apoptosis-induced cell death and confirmed that the elevated cytotoxicity observed for PEGSS-PTX/PTX is a result of the enhanced concentration of intracellular PTX. 3.5. In Vivo Antitumor Efficacy for PEG-SS-PTX/PTX NPs. The antitumor efficacy of the NP formulations was evaluated using a xenograft model of nude mice bearing MCF-7 tumors. According to the results of the pre-experiments, the dose of NPs was set to 7 mg/kg (calculated as the free-PTX dosage), and Taxol at the same dose was selected as a control. 3666

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Figure 8. In vivo antitumor efficacy of saline, Taxol (10 and 7 mg/kg), PEG-SS-PTX/PTX NPs (7 mg/kg), and PEG-PTX/PTX NPs (7 mg/kg) in MCF-7 tumor-bearing nude mice. (A) Tumor volume. (B) Tumor weight at the end of the test. (C) Body weight changes. (D) Photos of the excised tumors at the end of the test. Data represent the mean ± SD (n = 6). Red triangles indicate dose administration. *, p < 0.05 vs Taxol at the 7 mg/kg dose; ###, p < 0.001 vs PEG-PTX/PTX NPs. (E) Histopathological analysis by hematoxylin and eosin (H&E) staining of heart (a), liver (b), spleen (c), lung (d), and kidney (e) sections isolated from nude mice after treatment with saline or PEG-SS-PTX/PTX NPs.

that the PEG-SS-PTX/PTX NPs would show significantly better antitumor efficacy than that of the Genexol-PM formulation. 3.6. TUNEL Assay of Xenograft Tumors Treated with PEG-SS-PTX/PTX NPs. As shown in Figure 9, more obvious, extensive apoptotic tumor cells were found in the Taxol (10 mg/kg) and PEG-SS-PTX/PTX NP (7 mg/kg) groups than that in any other treatment groups. The apoptosis occurrence followed the order PEG-SS-PTX/PTX NPs (7 mg/kg) ≈ Taxol (10 mg/kg) > PEG-PTX/PTX NPs (7 mg/kg) > Taxol (7 mg/ kg) > saline group, which was in good agreement with the antitumor effects of various PTX formulations. These results

achieve both superior antitumor efficiency and lower toxicity, thus offering more advantages than those of Abraxane. PTXloaded polymeric micelles (e.g., Genexol-PM and NK105), formulated without introducing solvent and detergents, have a high MTD and lead to modest improvements in efficacy. However, in most cases, the micelles may not be effectively taken up by cells because of the stable PEG coating, and the payloads may not release sufficiently. In our previous study, PEG-PTX/PTX NPs showed greater activity in vivo than that of PTX-loaded monomethoxy-poly(ethylene glycol)-block-poly(D,L-lactide) (PEG-PLA/PTX) NPs, which was formulated in the same way as that of Genexol-PM,41 so it can be concluded 3667

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Figure 9. Apoptosis detected by TUNEL assay in xenograft tumors of mice treated with saline (a), Taxol (10 mg/kg) (b), Taxol (7 mg/kg) (c), PEG-SS-PTX/PTX NPs (7 mg/kg) (d), and PEG-PTX/PTX NPs (7 mg/kg) (e). Positive signals are revealed by fluorescein (green). Cell nuclei were stained with Hoechst 33258 (blue). Apoptotic cells are indicated by the colocalization of these two labels.

of free PTX and PTX conjugates, and antitumor effect assay after treatment with saline, Taxol, and PTX conjugates. This material is available free of charge via the Internet at http:// pubs.acs.org.

indicated that the redox-responsive PEG-SS-PTX/PTX NPs could deliver PTX to tumor tissues more efficiently and could consequently produce more severe cell apoptosis and tumor necrosis than either Taxol with the same dose or nonsensitive NPs. The redox-responsive NP delivery system that we developed here can achieve a high therapeutic index. Compared with other formulations of PTX, the advantages of PEG-SS-PTX/PTX NPs are that (1) the polymer−drug prodrugs covalently conjugated by disulfide bonds minimize the risk of drug leakage and preserve drug activity, (2) the self-assembly of free PTX and conjugated PTX guarantees a high drug content, (3) the stealth character of this system and the EPR effect decrease nonspecific tissue distribution, (4) the redox-triggered PEG corona detachment preserves drug activity, and (5) the programmed drug release enhances the overall concentration of the intracellular drug and prolongs the action time of the payload. The combination of these strategies contributes to the high anticancer efficiency and low incidence of side effects observed herein.

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Corresponding Author

*Tel: +86-10-82805935; Fax: +86-10-82805935; E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (nos. 81130059 and 81273456).

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REFERENCES

(1) Rowinsky, E. K.; Donehower, R. C. Paclitaxel (taxol). N. Engl. J. Med. 1995, 332, 1004−1014. (2) Onetto, N.; Canetta, R.; Winograd, B.; Catane, R.; Dougan, M.; Grechko, J.; et al. Overview of Taxol safety. J. Natl. Cancer Inst Monogr. 1993, 15, 131−139. (3) Duncan, R. Polymer conjugates as anticancer nanomedicines. Nat. Rev. Cancer. 2006, 6, 688−701. (4) Wang, Y.; Xin, D.; Liu, K.; Zhu, M.; Xiang, J. Heparin−paclitaxel conjugates as drug delivery system: synthesis, self-assembly property, drug release, and antitumor activity. Bioconjugate Chem. 2009, 20, 2214−2221. (5) Huang, Y.; Shi, J.; He, Q.; Sheikh, M. S. The promise of paclitaxel−peptide conjugates for MMP-2-targeted drug delivery. Cancer Biol. Ther. 2010, 9, 204−205. (6) Yang, D.; Liu, X.; Jiang, X.; Liu, Y.; Ying, W.; Wang, H.; et al. Effect of molecular weight of PGG−paclitaxel conjugates on in vitro and in vivo efficacy. J. Controlled Release 2012, 161, 124−131. (7) Harris, J. M.; Chess, R. B. Effect of PEGylation on pharmaceuticals. Nat. Rev. Drug Discovery 2003, 2, 214−221. (8) Pasut, G.; Veronese, F. M. PEG conjugates in clinical development or use as anticancer agents: an overview. Adv. Drug Delivery Rev. 2009, 61, 1177−1188. (9) Jokerst, J. V.; Lobovkina, T.; Zare, R. N.; Gambhir, S. S. Nanoparticle PEGylation for imaging and therapy. Nanomedicine 2011, 6, 715−728.

4. CONCLUSIONS In this study, we developed a novel PEG-SS-PTX/PTX NP delivery system featuring free PTX and conjugated PTX in one NP system with a redox-triggered, programmed drug-release mechanism. This system combined several benefits, including polymer−drug conjugates/prodrugs, high drug loading, the EPR effect, redox-specific PEG detachment, programmed drug release, lower toxicity, and enhanced antitumor efficacy. The data presented here demonstrate that these NPs possess a unique redox response and can effectively inhibit tumor growth both in vitro and in vivo. The potential of this drug system shows promise, and it may be further explored for use with various drugs for combination treatments or tumor-targeted drug delivery.

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AUTHOR INFORMATION

ASSOCIATED CONTENT

S Supporting Information *

Schematic illustrations of the structures of PEG-SS-MPA and PEG-SS-PTX, DSC thermograms of PEG-SS-PTX/PTX NPs, in vitro PTX release from PTX conjugates, in vitro cytotoxicity 3668

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