d-Î±-Tocopherol Polyethylene Glycol Succinate-Based Redox

Article pubs.acs.org/molecularpharmaceutics

D‑α-Tocopherol

Polyethylene Glycol Succinate-Based RedoxSensitive Paclitaxel Prodrug for Overcoming Multidrug Resistance in Cancer Cells Yuling Bao,†,§ Yuanyuan Guo,†,§ Xiangting Zhuang,† Dan Li,† Bolin Cheng,† Songwei Tan,*,†,‡ and Zhiping Zhang*,†,‡ †

Tongji School of Pharmacy, ‡National Engineering Research Center for Nanomedicine, Huazhong University of Science and Technology Wuhan 430030, P.R. China S Supporting Information *

ABSTRACT: To overcome the multidrug resistance (MDR) of P-glycoprotein (P-gp) substrate anticancer drugs, such as paclitaxel (PTX), a novel dual-functional prodrug, D-α-tocopherol polyethylene glycol succinate (TPGS) based PTX prodrug (TPGS-S-S-PTX), was synthesized here to fulfill the synergistic effect of P-gp inhibiting and intracellular redox-sensitive release. The prodrug could self-assemble into stable micelles in physiological environment with a diameter of ∼140 nm, while it disassociated in reductive condition and released PTX and TPGS active derivatives rapidly. High cell cytotoxicity in PTXresistant human ovarian cell line A2780/T was observed with enhanced PTX accumulation due to the P-gp inhibition by the TPGS moiety. The IC50 of TPGS-S-S-PTX was 55% and 91% more effective than that of Taxol (clinical formulation of PTX) and uncleavable TPGS-C-C-PTX prodrug, respectively. This was found to be related with the increased apoptosis/necrosis and cell arrest in G2/M phase. In vivo evaluation of the TPGS-S-S-PTX prodrug exhibited an extended half-life, increased AUC (area under the concentration−time curve), enhanced tumor distribution and significant tumor growth inhibition with reduced side effects as compared to Taxol and TPGS-C-C-PTX. This prodrug has great potential in improving efficiency in the treatment of MDR tumors. KEYWORDS: prodrug, redox-sensitive, TPGS, Paclitaxel, multidrug resistance HAMA−DOX17,18 have been reported, and some of them are undergoing clinical trials. These ester/amide-conjugated prodrugs can slowly and sustainably decompose into active drug in vivo and result in effective treatment in drug-sensitive tumor cells.19,20 However, the efficacy of these prodrugs may be limited in multidrug resistance (MDR) tumor cells. MDR is a common phenomenon in cancer cells which become resistant to the cytotoxicity of various structurally and mechanically unrelated chemotherapeutic agents. Among them, P-glycoprotein (P-gp) overexpression is an important mecha-

1. INTRODUCTION Nanotechnology-based drug delivery systems such as polymeric/inorganic nanoparticles, micelles, liposomes, nanogels, and prodrugs have been widely reported in anticancer applications.2−7 These have shown many advanced characteristics including increased drug solubility, extended circulation time by escaping from the reticuloendothelial (RES) system uptake, improved pharmacokinetic properties, enhanced therapeutic efficiency and reduced side effects, etc. Polymerbased prodrug has received much more attention due to the simple structure and great potential in clinical application.7−10 To date, many polymeric prodrugs, such as poly(ethylene glycol)−Paclitaxel (PEG−PTX),11 hydroxypropylmethacrylamide−PTX (HAMA−PTX),12 poly(L-glutamic acid)−PTX (PG−PTX), 1 3 −1 5 PEG−doxorubicin (PEG−DOX), 1 6 © 2014 American Chemical Society

Received: Revised: Accepted: Published: 3196

May 27, 2014 July 12, 2014 August 7, 2014 August 7, 2014 dx.doi.org/10.1021/mp500384d | Mol. Pharmaceutics 2014, 11, 3196−3209

Molecular Pharmaceutics

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nism, which can dramatically reduce intracellular drug concentration and thus limit the cytotoxic effects of drugs in tumors.21 To overcome the P-gp-based MDR, two strategies are mostly reported. One involves the design of a stimuliresponsive nanodrug, which responds to the tumor microenvironment and achieves an intracellular burst release of the active ingredients in tumor cells.22,23 The other employs the codelivery of the anticancer drug with P-gp inhibiting agent, e.g. tariquidar,24 verapamil,25 siRNA.26 In the case of polymeric prodrugs, some stimuli responsive polymeric prodrugs, such as PEG−poly(aspartate hydrazoneadriamycin),27,28 PEG-S−Scamptothecin (PEG-S-S-CPT),29 poly(ethylene oxide)−DOX (PEO−DOX)30 and dextran−PEI(-SS-DOX)31 have been studied, but only a few of them exhibited the possibility of reversing MDR. To the best of our knowledge, the application of the second strategy to overcome MDR by the use of prodrugs was seldom reported. A combination of both strategies may yield a smart novel prodrug that would be even more effective in reversing MDR. D-α-Tocopheryl polyethylene glycol succinate (TPGS) is a derivative of the natural vitamin E and PEG 1000. It has been approved by FDA as a safe pharmaceutic adjuvant and is used as solubilizer, absorption enhancer, and a vehicle for drug delivery systems.32,33 TPGS-based nanodrug delivery systems have exhibited enhanced drug encapsulation efficiency, cellular uptake, and cell cytotoxicity on cancer cells as well as an extended circulation time and enhanced therapeutic efficiency. More importantly, TPGS has shown great potential in overcoming MDR as a kind of P-gp inhibitor.34 Some TPGSrelated copolymers, such as PLA-TPGS,35 chitosan-g-TPGS,36 TPGS-b-poly(β-amino ester)37 and ditocopherol PEG2000 succinate,38 have been developed, and they possess the ability to overcome MDR. Some uncleavable TPGS-drug conjugations such as TPGS-DOX39,40 and TPGS-CPT41 have also been reported. However, the possibility of these prodrugs to reverse MDR was not shown. To develop a prodrug with both burst release and P-gp inhibition properties as mentioned above, a novel redoxsensitive PTX prodrug based on TPGS and disulfide bond was developed, and its ability to overcome MDR was investigated here. As we know, disulfide bonds are stable in human blood plasma. However, they can be cleaved under intracellular reductive conditions, where the glutathione (GSH) concentration is around 10 mM.1 This prodrug can be quickly cleaved after endocytosis by tumor cells to rapidly release the active drug and consequently achieve cancer cell cytotoxicity. The disassociated TPGS segment can bind with P-gp, restrain its activity and then reduce the efflux of PTX, so as to reverse MDR of cancer cells (Scheme 1). In vitro cytotoxicity and the mechanism of overcoming MDR was studied in PTX-sensitive human ovarian cell line A2780 and PTX-resistant human ovarian cell line A2780/T. In vivo pharmacokinetics, biodistribution, and the tumor inhibition effect were also evaluated in tumor-bearing mice models. Taxol (clinical formulation of PTX) and an uncleavable prodrug, TPGS-C-C-PTX with an insensitive conjugation bond, were chosen as the controls.

Scheme 1. Schematic Illustration of Redox-Sensitive TPGSS-S-PTX Prodrug for Overcoming MDR of Cancer Cells

pyridine (DMAP), rhodamine B (Rh-B) and rhodamine 123 (Rh-123) was purchased from Aladdin, China. 3,3′-Dithiodipropionic acid was obtained from TCI Shanghai, China. Penicillin−streptomycin, fetal bovine serum (FBS), RPMI 1640 medium, and trypsin without EDTA were purchased from Hyclone, U.S.A.. WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) was obtained from Roche, Germany. Hoechst 33342 was purchased from Biosharp, South Korea. All the solvents used were of analytical grade and were produced by Sinopharm, China. Annexin VFITC/PI double staining assay kit was supplied by KeyGEN, China. A2780 and A2780/T cell lines were supplied by KeyGEN, China. Mouse sarcoma tumor cell line S180 was provided by the Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences. Sprague-Dawley (SD) rats (200 ± 20 g) and Kunming mice (female, 5−7 weeks old, 18−20 g) were obtained from the Laboratory Animal Resources of Huazhong University of Science and Technology (Certificate No. SCXK 2010-0009). 2.2. Synthesis and Characterization of TPGS-PTX Prodrug. TPGS-S-S-PTX prodrug was synthesized by a twostep conjugation method. Dithiodipropionicanhydride (DTDPA) was synthesized first through a dehydration reaction.42,43 Then 3.08 g TPGS (2.0 mmol), 0.55 g DTDPA (3 mmol), and 0.25 g DMAP (2 mmol) were dissolved in 10 mL of anhydrous dimethylformamide (DMF) with 0.60 mL of triethylamine. The reaction was left to proceed for 24 h at room temperature. The products were dialyzed against ethanol (MWCO 2000) for 24 h, and dithiodipropionic TPGS ester (TPGS-S-S-COOH) was collected by reduced pressure distillation. The conjugation of PTX and TPGS-S-S-COOH was achieved by DCC/NHS method. Typically, TPGS-S-SCOOH (1.6 g, 1.0 mmol), NHS (0.14 g, 1.2 mmol), and DCC (0.20 g, 1.2 mol) were codissolved in anhydrous 5 mL DMF and reacted at room temperature for 24 h. Then the turbid liquid was filtered to remove the N,N-dicyclohexylurea (DCU) and mixed with a 5 mL solution containing 1.0 g PTX. After 48 h, the resulted prodrugs were purified by dialysis against DMF for 24 h (MWCO 2000) to remove unreacted impurities. Then DMF was replaced with ethanol. TPGS-S-S-PTX prodrug was finally collected by evaporating the ethanol. Uncleavable TPGS-C-C-PTX prodrug was prepared in a similar way except SA was used instead of DTDPA.

2. MATERIALS AND METHOD 2.1. Materials and Cells. PTX of purity 99% was obtained from Jinhe Limited, China. TPGS, succinic anhydride (SA), dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), RNase A and trypsin-EDTA were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). 4-dimethylamino 3197

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2.4. In Vitro Drug Release. The reduction-triggered release profiles of PTX from TPGS-PTX prodrug micelles were studied using a dialysis method (MWCO 2000) at 37 °C under sink conditions. A certain amount of freshly prepared TPGSPTX prodrug micelle solution was loaded into a dialysis bag (MWCO 2000) and dialyzed against 50 mL PBS (pH 7.4 or 5.5) in the presence or absence of 10 mM DTT or FBS at 37 °C in a water bath shaker at 110 rpm. At allocated time intervals, 10 mL of solution was removed followed by an addition of 10 mL fresh PBS. The drug concentration was determined by HPLC (Hitachi L-2000, Japan) equipped with a reverse phase Inertsil ODS-3 C18 column (150 × 4.6 mm, pore size 5 μm, Agilent, U.S.A.). 2.5. Cell Culture. A2780 and A2780/T cells were cultured in RPMI 1640 complete medium supplemented without/with 400 ng/mL Taxol in a humidified atmosphere incubator with 5% CO2 at 37 °C. After the cells grew to 80−90% confluence, they were trypsinized with 0.125% trypsin-EDTA (diluted in PBS). This was done every 2 days. 2.5.1. In Vitro Cellular Uptake of the TPGS-PTX Prodrug Micelles. Rh−B loaded TPGS-PTX prodrug micelles were used as a probe, and the cellular uptake was analyzed by confocal laser scanning microscopy (CLSM, Leica TCSNT1, Germany). Both A2780 and A2780/T cells were separately seeded onto 24-well plates which kept cover glass slips in 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 Rh−B loaded micelles at a concentration of 10 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 PBS and stained with Hoechst 33342 (10 μg/mL) for 8 min. The cells were then mounted on a glass slide for observation by CLSM. 2.5.2. Accumulation and Retention of PTX in MDR Cancer Cells. A2780/T cells were seeded in 6-well plates at a density of 1.0 × 105 cells/well and incubated overnight. The cells were washed with PBS and treated with free PTX or PTX-tethered prodrugs in culture medium with equivalent PTX at a concentration of 5 μg/mL. Cells were incubated at 37 °C for different periods of time (1, 6, 12, and 24 h) and washed twice with PBS; cells were lysed with lysis buffer (Beyotime, P0013B) containing PMSF. Lysates were centrifuged at 12000g at 4 °C for 10 min, and the supernatant was collected. PTX concentrations in cell lysates were measured by HPLC analyses and normalized to the total cellular protein content of the cells, which was determined by the BCA Protein Assay Kit (Beyotime, P0010). 2.5.3. Cytotoxicity Assay in Drug-Sensitive A2780 Cells and -Resistant A2780/T Cells. The in vitro cytotoxicity of TPGS-PTX prodrug micelles and Taxol was determined by WST-1 conversion assay in the following manner. A2780 and A2780/T cells in their logarithmic growth were seeded in 96well 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 treatment samples, including free PTX, TPGS-C-CPTX prodrug micelle, TPGS-S-S-PTX prodrug micelle and Taxol. The concentration of PTX used in each group was 0.025, 0.25, 2.5, 10, 25, 100 μg/mL, and the exposure duration was 24, 48, and 72 h, respectively. At designated time intervals, 10 μL of WST-1 solution was added to the wells for 30 min at 37 °C. The absorbance was measured at 490 nm using a

The prodrugs and intermediate products were characterized by 1HNMR spectra (Bruker AVANCE III 400 MHz NMR spectrometer, solvent: CDCl3) and Fourier transform infrared spectroscopy (FTIR, Bruker VERTEX 70 FTIR spectrophotometer). The molecular weight of the prodrugs and TPGS were detected by gel permeation chromatography (GPC, Waters-2410 system). The purity of the prodrugs was assayed by thin layer chromatography (TLC). 1 H NMR of TPGS-PTX (CDCl3, ppm): 0.86 (12H, −CH(CH3)CH3 and −CH2CH(CH3)CH2− TPGS), 1.00− 1.80 (−CH 2 CH 2 CH 2 CH(CH 3 )CH 2 CH 2 CH 2 CH(CH 3 )CH2CH2CH2CH(CH3)CH3 and −OCH(CH3)(CH2)− of TPGS), 1.16 (3H,C(15)−CH3 PTX), 1.23 (3H,C(15)−CH3 PTX), 1.69 (3H, C(11), CH3 PTX), 1.98−1.93 (2H PTX), 1.80 (3H, C(4)−OAc PTX), 2.01 (9H, PhCH3 TPGS), 2.22 (4H PTX), 2.36−2.29 (3H PTX), 2.39 (3H, C(4)−OAc PTX), 2.50−2.63 (2H, PhCH2CH2− TPGS, and 4H, SSCH2CH2COO− of DTDPA or CH2CH2COO− of SA), 2.80 (2H, −PhOCOCH2CH2COO− TPGS), 2.95 (2H −PhOCOCH2CH2COO− TPGS), 3.65 (92H, −OCHH2CH2O− TPGS), 4.25 (4H, −COOCH2CH2O− TPGS), 4.80 (1H, C(2′)−H PTX), 4.95 (1H, C(5)−H PTX), 5.42 (1H, C(7)−H PTX), 5.67 (1H, C(2)−H PTX), 5.80 (1H, C(3′)−H PTX), 6.19 (1H, C(13)−H PTX), 6.26 (1H, C(10)−H PTX), 7.03 (1H, NH PTX), 7.34−7.43 (5H PTX), 7.48−7.52 (5H PTX), 7.62(1H, p-OCH(O) Ph PTX), 7.75 (2H, o-NHC(O) Ph PTX), 8.15 (2H, o-C(O) Ph PTX). 2.3. Preparation and Characterization of TPGS-PTX Prodrug Micelles. TPGS-PTX (both TPGS-S-S-PTX and TPGS-C-C-PTX) prodrug micelles were prepared by a simple solvent-evaporation method. Briefly, 50 mg of prodrug was dissolved in 1.5 mL of ethanol. Then the solution was added into 15 mL PBS buffer (pH 7.4) or acetate buffer (pH 5.5) dropwise under magnetic stirring. The micelle solution was stirred overnight to remove the ethanol. The resulted mixture was filtered through 0.45 μm syringe filter. To prepare Rh−B-loaded TPGS-PTX prodrug micelles, 0.5 mg Rh−B was mixed with TPGS-PTX prodrug (50 mg) and codissolved in ethanol. The remaining steps were the same as in the preparation of TPGS-PTX prodrug micelle.The average particle size, size distribution, and ζ potential of the prodrug micelles were determined by the laser light scattering (DLS, Zeta Plus, Brookhaven, U.S.A.). Morphologies of the micelles were investigated by transmission electron microscope (TEM, JEM-1230, Japan). The critical micelle concentration (CMC) was determined by using fluorescence probe techniques.44 Steady-state fluorescence spectra were obtained on a Hitachi F4600 luminescence spectrometer. The prodrug micelle solutions with different concentrations were incubated with pyrene (6.0 × 10−7 M) overnight. Excitation spectra of the sample solutions were obtained at an emission wavelength of 372 nm with excitation spectra (300−350 nm). The change of the intensity ratio (I339/I333) of pyrene was plotted vs the sample concentration. The stability of the TPGS-PTX prodrug micelles under different environments was evaluated as follows: freshly prepared TPGS-S-S-PTX micelle solution was incubated at 37 °C in pH 7.4 PBS with or without the presence of 10 mM DTT. Time-dependent changes in micelle diameter were monitored for 8 h by DLS. At the conclusion of the test, the solutions were freeze-dried and analyzed by GPC and TCL. Parallel similar tests using uncleavable TPGS-C-C-PTX prodrug were also performed. 3198



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randomly divided into three groups (n = 3). The rats were injected intravenously with Taxol, TPGS-C-C-PTX prodrug micelle solution, and TPGS-S-S-PTX prodrug micelle solution at a dose of 10 mg PTX/kg, respectively. Blood samples were collected into EP tubes with heparin at 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 h for each group. The collected blood samples were centrifuged to obtain the plasma. Then the plasma was mixed with an equal volume of methanol and the mixture extracted with 1 mL methyl tertiary butyl ether (MTBE) and vortexed for 5 min. After centrifugation at 11,000 rpm for 5 min, the supernatant was collected and dried at 37 °C using N-EVAP (MTN-2800D, Autoscience, Tianjin, China). The solid residue was dissolved in a mixed solution (acetonitrile/water solution, 50/50, v/v) and filtered for analysis. The PTX concentrations were determined by HPLC. The standard curve is linear and ranges from 0.05 to 12.5 μg/mL (R2 = 0.9998). The analysis was operated by Agilent system with a Hypersil ODS column (5 μm, 4.6 mm × 150 mm) at 30 °C with the wavelength set at 227 nm. The mobile phase was acetonitrile and deionized water (50/50, v/v) at a rate of 1.0 mL/min. The pharmacokinetics parameters were calculated by the drug and statistics (DAS) software (version 2.1.1, Mathematical Pharmacology Professional Committee, China). 2.7. Biodistribution. To build S180 tumor-bearing mice models, Kunming mice were subcutaneously injected at the right forelimb axilla with 0.2 mL S180 cell suspension containing 1.0 × 107 cells. Then the tumor size in each mouse was measured daily by vernier calipers. The tumor volume was calculated as V = L × W2/2 (where W is width and L is length). The mice were randomly assigned to three groups (12 mice per group) after the tumor volume grew to 50−100 mm3. These were injected with Taxol, TPGS-C-C-PTX prodrug solution, and TPGS-S-S-PTX prodrug solution at a dose of 10 mg/kg PTX, respectively. At time intervals of 1, 6, 12, and 24 h after the injection, three mice in each group were sacrificed, and the tissues (heart, liver, spleen, lung, kidney and tumor) were collected. The tissues were then washed with saline, weighed, and homogenized with PBS. The PTX content was measured in a way similar to that used in the pharmacokinetics study. 2.8. Tumor Growth Inhibition. S180 tumor-bearing mice were prepared as in section 2.7. The mice were randomly assigned to four treatment groups (n = 5) after the tumor volume grew to 50−100 mm3 (denoted as day 1). Each group was treated by tail vein injection with saline, Taxol, TPGS-C-CPTX prodrug solution, and TPGS-S-S-PTX prodrug solution at a dose of 10 mg/kg PTX on day 1, 3, 5, and 7. The tumor sizes were measured every day to evaluate the antitumor efficiency. When the tumor length in the saline group increased to above 20 mm, all the mice were sacrificed, and the tumors were extirpated, weighed, and fixed in 4% paraformaldehyde. The inhibition ratio was calculated as follows:

microplate reader (Thermo, U.S.A.). IC50 (concentration resulting in 50% inhibition of cell growth) value was calculated by SPSS software (version 19.0). The experiment was repeated thrice. 2.5.4. Cell Apoptosis Analysis. Nuclear morphologies of A2780/T cells with different treatments were determined by Hoechst 33342 staining method. A2780/T cells were seeded into a 24-well plate which kept cover glass slips in each well, at a density of 1.0 × 104 cells and attached for 24 h. The cells were then incubated with medium containing Taxol or TPGS-PTX prodrug micelle at similar PTX concentration of 2.5 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 PBS and stained with 200 μL 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). Annexin V-FITC/PI double staining was performed to quantitatively analyze the apoptosis of A2780/T cells. The cells were seeded on the 6-well plates (1.0 × 105 cells/well) followed by treatment with Taxol, TPGS-C-C-PTX prodrug and TPGSS-S-PTX prodrug with the PTX concentration of 2.5 μg/mL for 24 h. Untreated cells were used as control. At the end of incubation period, the cells were trypsinized, collected, and resuspended in 300 μL of binding buffer. Thereafter, 3 μL of annexin V-FITC and 3 μL of PI were added and mixed for 30 min in the dark. The stained cells were analyzed using a flow cytometer (Becton Dickinson, San Jose, CA). Intracellular ATP level assay was also used to verify the apoptosis. A2780/T cells were seeded into 12-well plates (5.0 × 104 cells/well), incubated for 24 h at 37 °C, and then treated with Taxol, TPGS-C-C-PTX prodrug, and TPGS-S-S-PTX prodrug with the PTX concentration of 2.5 μg/mL for 24 h. Intracellular ATP levels were determined using the luciferin− luciferase-based ATP luminescence assay kit (Beyotime Institute of Biotechnology, China). 2.5.5. Cell Cycle Distribution Analysis. A2780/T cells were seeded into 6-well plates (1.0 × 105 cells/well) with Taxol or TPGS-PTX prodrug micelle (PTX concentration, 2.5 μg/mL). After 24 h 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 4 °C and stained with PI solution (50 μg/mL) for 30 min in the dark. The DNA content was measured by the FACS Calibur system (Becton Dickinson, San Jose, CA, U.S.A.), and the percentage of cells in each phase of the cell cycle was calculated using the ModFit software (Verity Software House, Topsham, ME, U.S.A.). 2.5.6. Inhibition of P-gp Efflux Test. Rh-123, a P-gp substrate fluorescent dye, is an index of assaying the transport activity of P-gp. A2780/T cells were seeded into 6-well plates (1.0 × 105 cells/well) with verapamil, TPGS-S-S-PTX, and TPGS-C-C-PTX and incubated overnight. They were then incubated with 5 μg/mL of Rh-123 for 30 min at 37 °C. At the end of incubation, the cells were washed with PBS thrice to remove free Rh-123 and kept in dye-free medium. The fluorescence intensity of Rh-123 in the cells was measured by FACS using ModFit software. 2.6. Pharmacokinetics Study. The pharmacokinetics of TPGS-S-S-PTX prodrug micelle, TPGS-C-C-PTX prodrug micelle, and Taxol was studied using SD rats. The rats were

inhibition ratio (%) = [(Ws − Wt)/Ws] × 100%

(where Ws is the average tumor weight of the saline group and Wt is that of the treatment group). The tumors were first cut into small histological sections. These were stained with hematoxylin and eosin (HE). Histological analyses were performed by light microscopy with a CAD system. 2.9. Statistical Analysis. Numerical data were expressed as mean ± standard deviation. The differences in the mean were 3199



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analyzed by one-way ANOVA using SPSS software (version 19.0). Statistical significance was set as p < 0.05.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of TPGS-PTX Prodrugs. The redox-sensitive TPGS-S-S-PTX prodrug and uncleavable TPGS-C-C-PTX prodrug were synthesized via a two-step esterification reaction, as shown in Scheme 2. DTDPA Scheme 2. Synthetic Route of TPGS-PTX Prodrugs

Figure 1. 1HNMR spectra characterization of TPGS-PTX prodrugs.

The structure of TPGS-C-C-PTX and TPGS-C-C-COOH was also investigated (Figure S1 in the SI). The newly appearing signals at 2.65−2.72 ppm in the 1H NMR spectrum of TPGS-C-C-COOH, which were assigned to the −CH2− CH2− part of succinyl group, confirmed the ring-opening reaction between SA and TPGS. The other characteristic signals of TPGS-C-C-COOH and TPGS-C-C-PTX were almost the same as TPGS-S-S-COOH and TPGS-S-S-PTX, which had been marked in the figures. GPC was further used to assess the TPGS-PTX prodrug (Figure S3 in the SI). The weight-average molecular weight (Mw) of TPGS was 2673 Da. After being conjugated with PTX, the Mw of TPGS-S-S-PTX and TPGS-C-C-PTX became 3245 and 3366 Da, respectively. The increase of Mw indicated that there was successful esterification between TPGS and PTX. The purity of TPGS-PTX prodrug was also examined by TLC (Figure S4 in the SI). The results proved that PTX was conjugated with TPGS and minimum free PTX remained. 3.2. Preparation and Characterization of TPGS-PTX Prodrug Micelles. TPGS-PTX prodrug micelles with ∼30% drug loading efficiency (DLE, defined in SI) could be prepared in a simple way using 5−10% ethanol as cosolvent, and the encap efficiency (EE) was 100%. However, for PTX-loaded TPGS micelle fabricated by this method, lots of PTX aggregated during the preparing process (Figure S5 in the SI). The DLE and EE were only 1.8% and 18%, respectively. The advantage of the TPGS-PTX prodrug is well presented here. The particle sizes of TPGS-S-S-PTX and TPGS-C-C-PTX prodrug micelle (Table 1 and Figure 2a) were 143.9 ± 13.7 and 134.5 ± 10.9 nm as measured by DLS, respectively, which were much bigger than that of TPGS micelle. This may be because the conjugation of PTX increased the hydrophobic content of the whole system and the hydrophobic−hydrophilic−hydro-

was first prepared and used instead of DTDP due to its high reactivity with the hydroxyl groups.42,43 As a result, the formation of the byproduct, TPGS-S-S-TPGS could be limited. The structure of TPGS-S-S-COOH was confirmed by 1H NMR (Figure S1 in the SI). The increase of the peak areas at 2.93 and 2.80 ppm, which belong to the two methylene groups of DTDPA, confirmed the successful reaction between TPGS and DTDPA. The increased intensity of 4.27 ppm also proved that the formation of an ester group had occurred. However, there were no obvious differences in the IR spectra (Figure S2b in the SI) of TPGS and TPGS-S-S-COOH except the slight enhancement of the peak at 1735 cm−1, which belonged to the stretching vibration of the CO bond (νCO) of the newborn ester bond. After the conjugation reaction of TPGS-SS-COOH and PTX, the 1HNMR spectrum (Figure 1) of the TPGS-S-S-PTX prodrug displayed all the expected resonance peaks characteristic of TPGS and PTX, such as the CH3 at 0.86 ppm, −CH2−CH2−O− at 3.65 ppm of TPGS and hydrogen in aromatic ring at 8.13 ppm of PTX, respectively. In the FTIR spectra (Figure S1a in the SI), the C−O stretching vibration (νC−O) of PEG at 1104 cm−1, which was the typical signal of TPGS and the C−H out-of-plane vibration at 715 cm−1, which stood for single substitution of the benzene ring in PTX, could be both found. The 1H NMR and FTIR results indicated the successful synthesis of TPGS-S-S-PTX prodrug.

Table 1. Characterization of TPGS, TPGS-C-C-PTX and TPGS-S-S-PTX Micelles

a

3200

micelles

diameter (nm)a

ζ-potential (mV)a

CMC (g/L)b

TPGS TPGS-C-C-PTX TPGS-S-S-PTX

14.0 ± 3.7 134.5 ± 10.9 143.9 ± 13.7

−4.8 ± 0.8 −8.1 ± 1.4 −8.5 ± 0.5

2.5 × 10−3 4.5 × 10−3 1.0 × 10−3

Measured by DLS. bDetermined using fluorescence probe technique. dx.doi.org/10.1021/mp500384d | Mol. Pharmaceutics 2014, 11, 3196−3209


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Figure 2. Characterization of TPGS-PTX prodrug micelles. (a) DLS result, (b) TEM image of TPGS-S-S-PTX prodrug micelles, (c) plot of the intensity ratio I339/I333 as a function of logC for TPGS, TPGS-C-C-PTX prodrug micelle, TPGS-S-S-PTX prodrug micelle, [Py] = 6.0 × 10−7 M, (d) change in the diameter of TPGS-C-C-PTX and TPGS-S-S-PTX prodrug micelles at pH 7.4 with or without 10 mM DTT, (e) in vitro release of PTX from TPGS-S-S-PTX (solid) and TPGS-C-C-PTX (dash) prodrug at pH 7.4 or 5.5 in the presence or absence of 10 mM DTT.

phobic triblock structure of TPGS-PTX limited the free volume of PEG segment. The ζ-potentials of TPGS-S-S-PTX and TPGS-C-C-PTX prodrug micelle were −8.5 and −8.1 mV, respectively, which was slightly higher thanthat of TPGS micelle (−4.8 mV). This may be caused by the different PEG density on the micelle surface. TEM was further used to confirm the micelle structure of TPGS-S-S-PTX and TPGS-CC-PTX prodrug micelles (Figure 2b). The micelles had uniform spherical shapes with diameters about 80 nm. The CMC values of TPGS-S-S-PTX and TPGS-C-C-PTX prodrug micelles were determined by pyrene probe fluorescence technology (Figure 2c and Table 1). The CMC of TPGS was 2.5 × 10−3 g/L. This is close to previous research but lower than the value obtained through surface tension (0.02 wt %) due to the different determination methods applied.45 The CMC value of TPGS-S-S-PTX prodrug micelle was 60% lower than that of TPGS, while that of TPGS-C-C-PTX prodrug micelle was much higher. This may be due to the different linkers between TPGS and PTX.46 3.3. Redox-Sensitive Structure Change of TPGS-PTX Prodrug Micelles. The time-dependent size change of TPGSPTX prodrug micelles under different conditions (with/without 10 mM DTT) was investigated by DLS to present the redoxsensitive destabilization of TPGS-S-S-PTX prodrug micelles

(Figure 2d). The micelles were quite stable at both pH 7.4 and 5.5 for more than 48 h (data not shown). However, an increase in particle size was observed in the presence of 10 mM DTT, and the average diameter doubled within 8 h, which indicated the cleavage of the disulfide bond. This hypothesis was confirmed by GPC and TLC (Figures S3 and S4 in the SI). The GPC spectrum of TPGS-S-S-PTX prodrug changed from a single peak to two peaks with the Mp (molecular weight at peak maximum) of 2672 and 3083 Da after 8 h incubation with 10 mM DTT. The Mw of TPGS-S-S-PTX prodrug changed to 2734 Da in the end, which was almost the same as that of TPGS. The molecular weight decrease of TPGS-S-S-PTX prodrug confirmed the loss of PTX by cleavage of the disulfide bond. In consistency with GPC results, the TLC tests showed that the TPGS-S-S-PTX prodrug dissociated into TPGS and PTX after incubating with 10 mM DTT. All the results suggested that TPGS-S-S-PTX prodrug was able to achieve a dissociation process in the intracellular reductive microenvironment. In contrast, TPGS-C-C-PTX prodrug micelle was not very stable and slightly aggregated at pH 7.4. This may be caused by its relatively high CMC value. 3.4. In Vitro Reduction-Triggered Drug Release. It is well-known that there exists a reductive condition in tumor cells. Accordingly, the release of PTX from the TPGS-S-S-PTX 3201



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Figure 3. CLSM images of A2780 and A2780/T incubated with (a) Rh−B, (b) Rh−B-loaded TPGS-C-C-PTX prodrug micelle and (c) Rh−Bloaded TPGS-S-S-PTX prodrug micelle (red) for 2 h. The cells were fixed and stained with Hoechst 33342.

prodrug micelles would be accelerated. The drug release behavior of the prodrug micelles was processed in the presence or absence of 10 mM DTT under different pHs to investigate the reductive-trigged drug release mechanism, as shown in Figure 2e. A burst release was observed, and about 70% of PTX was released at pH 7.4 (corresponds to normal physiological environment) and pH 5.5 (simulates the pH in endosomes of tumor cells) after incubation with DTT for 12 h. In contrast, only 18% of PTX was released from TPGS-S-S-PTX prodrug micelles at pH 7.4 for the first 12 h under nonreductive conditions. At pH 5.5, the release rate was slightly accelerated because of the higher hydrolysis speed of the ester bond under acidic condition, and 29% of PTX was released at 12 h. The cumulative release of PTX increased to ∼90% at 120 h with the presence of 10 mM DTT at both pH 7.4 and 5.5, while the values were 23% and 54% without the DTT at pH 7.4 and 5.5, respectively. The TPGS-S-S-PTX prodrug also showed high stability in FBS and only 25% of PTX was released during the 120 h test. Thus, we can conclude that the drug release of TPGS-S-S-PTX prodrug micelles was suppressed in normal physiological environment but accelerated under the intracellular reducing microenvironment (both endosome and cytoplasm){Brü lisauer, 2014 #51}. For TPGS-C-C-PTX prodrug, the release ratio was independent of the DTT concentration due to the uncleavable -C−C- bond. 3.5. Cellular Uptake of TPGS-PTX Prodrug Micelles. Rh−B was used as a probe to test the ability of the cellular uptake of TPGS-PTX prodrug micelles. The cellular uptake was performed by CLSM in the A2780 and A2780/T cell lines. As shown in Figure 3, after 2 h of exposure, all samples exhibited significant red fluorescence throughout the cytoplasm, which is closely located around the nuclei (blue) in A2780 cells. This suggested that Rh−B and Rh−B-loaded prodrug micelles had been effectively taken up by the cells. However, it is hard for Rh−B to accumulate in cytoplasm of drug-resistant A2780/T cells since it is a kind of P-gp substrate.47,48 Only with the help of TPGS-PTX prodrug micelles does Rh−B achieve significant distribution in the cytoplasm. In order to further demonstrate the free PTX accumulation and retention in A2780/T cells via the redox-responsive prodrug formulation, the intracellular PTX accumulation was quantitatively determined at different time intervals 1, 6, 12, and 24 h. Figure 4 illustrates a much higher intracellular

Figure 4. PTX accumulation in A2780/T cells after incubation with Taxol, TPGS-C-C-PTX prodrug, and TPGS-S-S-PTX prodrug for different periods of time. The concentration of PTX (free PTX or equivalent) in cell culture was 5 μg/mL in all the experiments.

accumulation of free PTX in A2780/T cells for TPGS-S-S-PTX compared with Taxol and TPGS-C-C-PTX. The intracellular PTX amount increased rapidly with the extension of culturing time, reaching 2.75 ± 0.09 and 3.06 ± 0.21 μg per mg protein at 12 h and 24 h, respectively, which was about 3 times higher than the value after Taxol treatment. In contrast, due to the relative low hydrolysis rate of the ester bond in TPGS-C-CPTX prodrug, the intracellular free PTX stayed at a low level. These results confirmed that the quick cleavage of “S−S” bonds leads to an increased intracellular free PTX amount as supposed. This was consistent with the CLSM result. 3.6. In Vitro Cytotoxicity of TPGS-PTX Prodrug. To determine the cytotoxicity of TPGS-PTX prodrug micelles, in vitro cell growth inhibition experiments were performed on both A2780 and A2780/T cells (Figure 5). All the samples exhibited significant cell cytotoxicity dependent on drug concentration and incubation time against drug-sensitive A2780 cells. For the free PTX, the cell cytotoxicity was not as notable as that for the other three due to the low solubility of PTX. With the help of Cremophor EL, the cell growth inhibition ability of clinically used PTX, Taxol, was greatly improved. The viability in the Taxol group was much lower than that of the PTX group. Considering that Taxol is a clinical formulation with Cremophor EL (polyoxyethylatede castor oil) 3202



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Figure 5. In vitro cytotoxicity of PTX, TPGS-C-C-PTX, TPGS-S-S-PTX, and Taxol against A2780 and A2780/T cells after treatment for 24, 48, and 72 h. *, p < 0.05.

h with PTX concentrations as high as 100 μg/mL. The cytotoxicity of Taxol was also weakened obviously even though Cremophor EL had also been reported as a kind of P-gp inhibitor. As expected, TPGS-S-S-PTX was the most effective drug in cytotoxicity studies. For example, after 48 h incubation at a PTX concentration of 25 μg/mL, the cell viability of TPGS-C-C-PTX, TPGS-S-S-PTX, and Taxol was 41.3%, 13.3%, and 26.6%, respectively. This showed a 50.0% and 67.8% increase by TPGS-S-S-PTX as compared with that by Taxol and TPGS-C-C-PTX (p < 0.05). In consistency with the cytotoxicity results, the IC50 of TPGS-S-S-PTX was the lowest among the samples. It decreased by 54.7%, 98.1% at 24 h, 59.5%, 85.7% at 48 h and 52.0%, 86.5% at 72 h compared with Taxol and TPGS-C-C-PTX, respectively. The resistance index (RI), defined as IC50 (A2780/T)/IC50 (A2780), was calculated as 29.30, 29.05, 7.53 at 24 h, 219.00, 206.67, 88.67 at 48 h and 274.00, 324.50, 131.50 at 72 h for Taxol, TPGS-C-C-PTX, and TPGS-S-S-PTX, respectively. TPGS-S-S-PTX exhibited the strongest ability in reversing drug resistance. 3.7. Cell Apoptosis Assays. The apoptosis-inducing ability of TPGS-PTX prodrugs against MDR A2780/T cells was first qualitatively evaluated by Hoechst staining and observed by fluorescence microscopy. As shown in Figure 6a, the nuclei in the control were homogeneously stained in a manner similar to the cytoplasm. Some typical apoptotic features, such as cell shrinkage, chromatin condensation, fragmentation of the

as adjuvant which enhanced the solubility of PTX and widely used as a positive control to evaluate novel PTX formulations in vitro and in vivo investigations, we chose Taxol as the positive control in the following experiments. For TPGS-C-CPTX and TPGS-S-S-PTX, the cell cytotoxicity was significantly different after incubating for 24 h, while they were almost similar after 48 and 72 h incubation. This might be attributable to the different release ratios of PTX from the formulations. Although some researches found that certain intracellular environments can be obstacles to the cleavage of disulfidebased drug conjugates,49 in our study, the quick PTX release from TPGS-S-S-PTX in response to the reduction environment may still work because its cytotoxicity was much higher than that of nonredox-sensitive TPGS-C-C-PTX and to some degree, even equal to Taxol within the first 24 of the 72 h test. But the total PTX amount hydrolyzed from TPGS-C-CPTX could accumulate with time and also kill the cells effectively. IC50 (Figure 5) was further calculated to evaluate the cell cytotoxicities of Taxol, TPGS-S-S-PTX, and TPGS-CC-PTX. As much as the IC50 of Taxol was most effective during the first 24 h, it showed no difference at 48 and 72 h for Taxol, TPGS-S-S-PTX, and TPGS-C-C-PTX. Both the prodrugs were effective in treating non-MDR cancer. When it came to drug-resistant A2780/T cells, the results were quite different. Due to the drug-resistant nature, the free PTX showed minimal cytotoxicity even after incubating for 72 3203



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Figure 6. Cell apoptosis analysis and cycle distribution of A2780/T cells with Taxol, TPGS-C-C-PTX, and TPGS-S-S-PTX after 24 h treatment. (a) Nucleus apoptosis assay. (b) Annexin V-FITC/PI double staining by flow cytometry. (c) Intracellular ATP levels. (d) Cell cycle distribution.

PI negative) of TPGS-C-C-PTX (6.6%) was a little higher than that of Taxol (5.4%) and TPGS-S-S-PTX (3.6%). The apoptosis distribution tendency was similar to a recently reported MMP2-sensitive PEG-PTX prodrug, but the reason for this increased necrotic/dead cells amount is not clear.50 To further confirm the cell apoptosis result, ATPase assay was performed (Figure 6c). Cell apoptosis/necrosis is usually accompanied by reduction of intracellular ATP levels.51 The intracellular ATP levels of Taxol, TPGS-S-S-PTX prodrug, and TPGS-C-C-PTX prodrug were 75.0 ± 7.8%, 68.4 ± 7.1% and 81.2 ± 6.8%, respectively, compared with the control. The reduced ATP content was consistent with the qualitative and quantitative apoptosis results. All the results proved that TPGSS-S-PTX was the most effective prodrug in apoptosis experiments. This may be attributed to the drug release ratio and P-gp inhibition activity.

nucleus, and the appearance of apoptotic body could be observed in the cells treated with TPGS-C-C-PTX and Taxol. The number of apoptotic cells was also increased when they were treated with TPGS-S-S-PTX. This was in harmony with the results of MTT assay. In order to quantitatively verify the extent of apoptosis caused by TPGS-PTX prodrugs, annexin V/ FITC-PI staining assay was performed and analyzed (Figure 6b). The percentage of viable cells (Q3, both annexin and PI negative) for Taxol, TPGS-C-C-PTX prodrug and TPGS-S-SPTX prodrug were 78.5%, 88.1%, and 72.5%, respectively, which was much lower than that of untreated cells (100%). The percentage of late apoptotic cells (Q2, both annexin and PI positive) of Taxol and TPGS-S-S-PTX was almost similar (9.9% vs 10.9%), while the necrotic/dead cells (Q1, PI positive and annexin negative) for TPGS-S-S-PTX (13.0%) was much higher than that of Taxol (6.2%). It was also established that the amount of early apoptotic cells (Q4, annexin positive and 3204



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3.8. Cell Cycle Arrest. PTX has the property of inhibiting cell division and inducing cell cycle arrest in G2/M phase. The effects of Taxol, TPGS-C-C-PTX prodrug, and TPGS-S-S-PTX prodrug on cell cycle progression against A2780/T cells were analyzed by flow cytometry. According to Figure 6d, the percentage of cells in G2/M phase increased to 63.24%, 40.25%, and 78.14% for Taxol, TPGS-C-C-PTX prodrug, and TPGS-S-S-PTX prodrug, respectively, which was remarkably higher than the control (14.50%) after 24 h of exposure. The rate of the ability to induce cell cycle arrest in G2/M phase was TPGS-S-S-PTX > Taxol > TPGS-C-C-PTX, which was consistent with that of the cell apoptosis experiments. The differences between TPGS-C-C-PTX prodrug and TPGS-S-SPTX prodrug further proved the advantage of the redoxresponsive property. 3.9. P-gp Inhibition Assays. Overexpressed P-gp on the membrane of A2780/T cells was one of the main causes of the MDR phenotype.23 P-gp expression level of A2780 and A2780/ T cells used in this work was first detected by Western blot (Figure S6 in the SI). A2780/T cells exhibited significant P-gp overexpression compared with A2780 cells. Rh-123 retention fluorescent intensity was used to evaluate the effect of TPGSPTX prodrug on P-gp. As seen in Figure 7, the fluorescence

tail vein injection. The plasma concentration−time profiles are shown in Figure 8, and the corresponding PK parameters are

Figure 8. Pharmacokinetics behavior after intravenous injection to SD rats of Taxol, TPGS-C-C-PTX prodrug, and TPGS-S-S-PTX prodrug at a dose of 10 mg PTX/kg. (n = 3).

Table 2. Pharmacokinetic Parameters of Taxol and TPGS-CC-PTX and TPGS-S-S-PTX Prodrugs in Rat after Intravenous Injection at an Equivalent Dose of 10 mg/kg (n = 3)

Figure 7. Rh123 intracellular accumulation in A2780/T cells treated with verapamil, TPGS-C-C-PTX prodrug, and TPGS-S-S-PTX prodrug after 24 h incubation.

parameters

unit

AUC0−t AUC0−∞ MRT0−t MRT0−∞ t1/2 CL V Tmax Cmax

mg/L·h mg/L·h h h h L/h/kg L/kg h mg/L

TPGS-C-CPTX

taxol 23.45 23.55 1.75 1.81 2.22 0.43 1.37 0.50 11.38

± ± ± ± ± ± ±

1.93 1.78 0.13 0.44 0.20 0.03 0.23

± 0.62

12.08 12.20 2.63 2.96 4.02 0.82 4.90 0.50 5.14

± ± ± ± ± ± ±

1.20 1.06 0.20 0.58 2.15 0.07 2.94

± 0.24

TPGS-S-SPTX 40.99 41.18 9.11 9.47 9.43 0.25 3.34 0.50 7.03

± ± ± ± ± ± ±

6.48 6.60 0.17 0.31 0.40 0.04 0.34

± 0.71

summarized in Table 2. All plasma profiles were found to be in line with the two-compartment model. Taxol was rapidly removed from the circulatory system. The blood circulation time of TPGS-S-S-PTX prodrug was significantly extended compared with that of Taxol, with a 4.25 times longer t1/2, 5.23 times higher MRT0∼t, 1.75 times larger area under the curve (AUC0∼t), and substantially lower values of clearance (CL, 58.0%). However, TPGS-C-C-PTX prodrug did not perform well, as compared to TPGS-S-S-PTX prodrug. Its t1/2 and MRT0∼t were 1.81- and 1.51-fold of that of Taxol, but it showed lower AUC0∼t and higher CL. On the other hand, a toxic concentration was achieved in the first 45 min after injection of Taxol (above 8.54 μg/mL32). For TPGS-C-C-PTX and TPGSS-S-PTX prodrug, the maximum concentration tested was 7.03 μg/mL and 5.14 μg/mL, respectively. Both the TPGS-PTX prodrugs could circumvent the systemic toxicity caused by high doses of PTX. 3.11. Biodistribution of TPGS-PTX Prodrug. To evaluate the passive targeting of TPGS-PTX prodrugs to S180 xenograft tumor, the tissue distribution of PTX with the administration of prodrugs and Taxol was investigated in tissues including heart,

intensity of Rh-123 of the control was quite low because Rh123 is a kind P-gp substrate, which could easily be pumped out by A2780/T cells. Increased fluorescence intensity was observed in the cells treated with the verapamil and TPGSPTX prodrugs, which indicated that the function of P-gp efflux pump was weakened. The intensity of TPGS-S-S-PTX prodrug was much higher than that of TPGS-C-C-PTX prodrug. This indicated that the P-gp inhibition ability of free TPGS dissociated from TPGS-S-S-PTX was stronger than TPGS-CC-PTX. It was worth noting that both the two prodrugs were more effective than verapamil, a clinically used P-gp inhibitor. Therefore, it might be concluded that TPGS-based prodrug can inhibit the function of P-gp, prevent the “pumping-out” of the drug, and thus result in an increase of chemosensitivity on A2780/T cells. 3.10. Pharmacokinetics Study of TPGS-PTX Prodrug. The pharmacokinetics of TPGS-C-C-PTX prodrug, TPGS-S-SPTX prodrug, and Taxol were studied by administering the respective prodrugs to SD rats at a dose of 10 mg PTX/kg by 3205



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Taxol, which indicated that the prodrug formulation could reduce the cardiotoxicity and renal toxicity of PTX. The biodistribution tendency of TPGS-C-C-PTX was close to that of TPGS-S-S-PTX besides the increased liver and kidney accumulation and weak tumor targeting ability. This may be caused by the instability of TPGS-C-C-PTX micelle due to its relative higher CMC value. Large particles were easily formed after injection and thus resulted in a quick elimination from the body. 3.12. In Vivo Antitumor Activity of TPGS-PTX Prodrug. The in vivo antitumor efficiency of TPGS-PTX prodrugs and Taxol was further evaluated in S180-tumor-bearing mice. The results are detailed in Figure 10a. The body weight of the four

liver, spleen, lung, kidney, and tumor at different time points (Figure 9). The biodistribution of PTX was significantly varied

Figure 10. In vivo antitumor efficacy in S180 tumor-bearing Kunming mice treated with Taxol, TPGS-C-C-PTX prodrug, and TPGS-S-SPTX prodrug at a dose of 10 mg PTX/kg (n = 5). (a) Relative tumor growth ratio, (b) HE staining assay of the tumor sections.

groups increased, indicating that there was no significant toxicity for all the formulations. The changes in tumor volume showed that both TPGS-S-S-PTX prodrug and Taxol were able to inhibit the tumor growth effectively. The therapeutic efficacy of the TPGS-S-S-PTX prodrug was the best while TPGS-C-CPTX prodrug showed little effect. The tumor inhibition rates of Taxol, TPGS-C-C-PTX prodrug, and TPGS-S-S-PTX prodrug were 45.31%, 6.15%, and 52.43%, respectively (Table S1 in the SI). The results were in accordance with the tendency of cell cytotoxicity and pharmacokinetics. The tumor growth inhibition on Balb/c mice-bearing MCF-7 cells (human breast cancer cells) also exhibited a similar tendency (data not shown). To further evaluate the antitumor effect of TPGS-PTX prodrugs, the tumors were excised for pathology. Figure 10b is the representative tumor sections from different mice groups. The histopathology of tumor from each group was observed by HE staining. In the saline group, the tumor cells were polykaryocytes with large irregular karyons and rich cytoplasm and more nuclear division. The groups injected with drug showed spotty necrosis and nuclear fission in the tumor section. Significant spotty necrosis and intercellular blank were observed in the tumor tissue treated with TPGS-S-S-PTX

Figure 9. In vivo biodistribution of (a) Taxol, (b) TPGS-C-C-PTX prodrug, and (c) TPGS-S-S-PTX prodrug after intravenous injection at a dose of 10 mg PTX/kg to S180 tumor-bearing Kunming mice at 1, 6, 12, and 24 h. (n = 3).

by the TPGS-PTX prodrug formulations. TPGS-S-S-PTX prodrug showed significantly enhanced tumor accumulation compared with Taxol. At 1 h time point, the PTX concentration was ∼7 μg/g, which was roughly equivalent to that of Taxol. Afterward, its amount of PTX in the tumor was 1.5, 1.8, and 2.6 times higher than that of Taxol at 6, 12, and 24 h, respectively. This was due to the long circulation time and EPR effect of TPGS-S-S-PTX prodrug micelle. The liver and spleen distribution of TPGS-S-S-PTX prodrug was slightly higher than that of Taxol. The reason may be that the TPGS on the micelle surface increased the phagocytosis by macrophages.39,40The amount of PTX in the TPGS-S-S-PTX group in heart and kidney were obviously decreased as compared to 3206



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data of in vivo tumor inhibition experiment. This material is available free of charge via the Internet at http://pubs.acs.org.

prodrug and Taxol, especially with TPGS-S-S-PTX prodrug, while there were only some spherical cells in the TPGS-C-CPTX prodrug group. All results confirmed that TPGS-S-S-PTX prodrug is the most effective tumor-inhibiting product compared to the others.

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*Fax and phone: +86-27-83601832. E-mail: zhipingzhang@ mail.hust.edu.cn (Z.P. Zhang). *E-mail [email protected] (S.W. Tan).

4. CONCLUSION A novel kind of dual-function redox-sensitive prodrug, TPGS-SS-PTX, was successfully synthesized and applied in overcoming MDR. The prodrug could self-assemble into micelles, followed by enrichment in tumor through the EPR effect. The disulfide bond would disassociate in the cancer cells, which resulted in the rapid release of PTX and TPGS active ingredient. P-gp could be blocked by TPGS and intracellular PTX concentration was improved. Hence, increased cell cytotoxicity and higher cell apoptosis/necrosis against MDR cells were observed compared to Taxol and uncleavable TPGS-C-C-PTX prodrug. In vivo evaluation also demonstrated the advantages of TPGS-S-S-PTX prodrug which included better pharmacokinetic properties, higher tumor accumulation, lower systemic cytotoxicity, and stronger tumor growth inhibition. The significant differences between TPGS-C-C-PTX and TPGS-S-S-PTX are intriguing. Despite very similar structures, the two conjugates exhibit different physical characteristics, stability, IC50, and pharmacokinetics. As we assumed, after the conjugation of PTX, a hydrophobic−hydrophilic−hydrophobic triblock structure was formed in TPGS-PTX. Thus, the structure of TPGS-PTX micelle was “flower-like” but not “star-like”.52 A short and rigid linker between TPGS and PTX will limit the free volume of the PEG segment and affect the topological structure of the micelle. So the micelle stability of TPGS-C-C-PTX with only two carbon linkers was found to be lower than that of TPGS-S-SPTX with a four carbons and two sulfurs soft linker.46 This resulted in a relatively higher CMC value and instability, which means that the TPGS-C-C-PTX micelle would easily aggregate during the circulation and be eliminated from the body, as shown in Figure 2c,d and Figure 8. Moreover, PTX release ratio from TPGS-C-C-PTX is quite slow due to the insensitive “−CC−” conjugation (Figure 2e). These may be the key reason why there is a crucial difference dynamic characteristic between TPGS-C-C-PTX and TPGS-S-S-PTX in vivo and in vitro. Even though TPGS-S-S-PTX prodrug exhibited a satisfactory treatment effect and the PTX content of TPGS-S-S-PTX in tumor was 1−2 times more than that of Taxol (Figure 9), its accumulation in tumor was unremarkable. The enrichment of micelles in tumor was based on the EPR effect. However, several recent papers have questioned this presumption as rarely operative in humans, and rarely proven in mice.53−55 Using targeting group-modified drug delivery systems, such as RGD ligand, may improve the tumor accumulation as well as the efficacy of cancer treatment. Our future work will focus on this concept to perfect this prodrug system. Taking into consideration the limitations, we still believe that this simple but effective prodrug will provide a new platform for treating MDR solid tumors.

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AUTHOR INFORMATION

Corresponding Authors

Author Contributions §

Y.L. Bao and Y.Y. Guo contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported by National Basic Research Program of China (973 Program, 2012CB932501), the National Nature Science Fund of China (21204024 and 81373360), Doctoral Fund of Ministry of Education of China (20120142120093), China Postdoctoral Science Foundation funded project (2013T60722), the Fundamental Research Funds for the Central Universities (2014TS091 and 2014QN134), Chutian Scholar Award and 2013 Youth Scholar Award of HUST, Students Research Fund of HUST.

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ABBREVIATIONS P-gp,P-glycoprotein; MDR,multidrug resistance; TPGS,D-αtocopheryl polyethylene glycol 1000; PTX,Paclitaxel; Taxol,clinical formulation of PTX, with 50:50 Cremophor EL and alcohol; IC50,concentration resulting in 50% inhibition of cell growth; RES,reticuloendothelial; PEG,poly(ethylene glycol); HAMA,hydroxypropylmethacrylamide; PG,poly(L-glutamic acid); PEO,poly(ethylene oxide); GSH,glutathione; SA,succinic anhydride; DCC,dicyclohexylcarbodiimide; NHS,N-hydroxysuccinimide; PI,propidium iodide; DMAP,4-dimethylamino pyridine; Rh,rhodamine; WST-1,4-[3-(4-iodophenyl)-2-(4-nitrop-henyl)-2H-5-tetrazolio]-1,3-benzene disulfonate; FBS,fetal bovine serum; ATP,adenosine triphosphate; AUC,area under the concentration−time curve; MRT,mean residence time

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The details and results of Western blot analysis, the preparation and characterization of PTX-loaded TPGS micelles, 1H NMR, FTIR, GPC spectra and thin layer chromatography results of rough materials, intermediate or degraded products, additional 3207



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d-Î±-Tocopherol Polyethylene Glycol Succinate-Based Redox

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