Nitric Oxide Releasing d-α-Tocopheryl Polyethylene Glycol Succinate

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Nitric Oxide Releasing D‑α-Tocopheryl Polyethylene Glycol Succinate for Enhancing Antitumor Activity of Doxorubicin Qingle Song,† Songwei Tan,†,‡ Xiangting Zhuang,† Yuanyuan Guo,† Yongdan Zhao,† Tingting Wu,† Qi Ye,† Luqin Si,*,† and Zhiping Zhang*,†,‡ †

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

ABSTRACT: Nitric oxide (NO) has attracted much attention for its antitumor activity and synergistic effects when codelivered with anticancer agents. However, due to its chemical instability and short half-life, delivering gaseous NO directly to tumors is still challenging. Herein, we synthesized a NO releasing polymer, nitrate functionalized D-α-tocopheryl polyethylene glycol succinate (TNO3). TNO3 was able to self-assemble into stable micelles in physiological conditions, accumulate in tumors, and release ∼90% of NO content in cancer cells for 96 h. It further exhibited significant cancer cell cytotoxicity and apoptosis compared with nitroglycerine (GTN). Notably, TNO3 could also serve as an enhancer for the common chemotherapeutic drug doxorubicin (DOX). Codelivering TNO3 with DOX to hepatocarcinoma HepG2 cancer cells strengthened the cellular uptake of DOX and enabled the synergistic effect between NO and DOX to induce higher cytotoxicity (∼6.25-fold lower IC50). Moreover, for DOX-based chemotherapy in tumor-bearing mice, coadministration with TNO3 significantly extended the blood circulation time of DOX (14.7-fold t1/2, 6.5-fold mean residence time (MRT), and 13.7-fold area under curve (AUC)) and enhanced its tumor accumulation and penetration, thus resulting in better antitumor efficacy. In summary, this new NO donor, TNO3, may provide a simple but effective strategy to enhance the therapeutic efficacy of chemotherapeutic drugs. KEYWORDS: nitric oxide, TPGS, doxorubicin, antitumor et al.20 developed S-nitrosoglutathione (GSNO) conjugated POEGMA-b-PVDM micelles, which dramatically improved the stability of GSNO, and extended its half-life to over 14 days. Che et al.21 synthesized ruthenium nanoparticles with tert-butyl nitrite (tBuONO) to deliver NO in aqueous media. D-α-Tocopheryl polyethylene glycol succinate (TPGS) is a water-soluble derivative of natural vitamin E, which has amphiphilic structure comprising lipophilic tail and hydrophilic polar head portion.22 It has been approved by FDA as a pharmaceutical ingredient. TPGS has a low critical micelle concentration (CMC) of 0.02% w/w and high stability in the blood.23 In recent years, it has been intensively applied in drug delivery systems as an absorption enhancer, emulsifier, solubilizer, and stabilizer.24,25 In addition, TPGS-based nano drug delivery systems have exhibited enhanced cellular uptake and cancer cell cytotoxicity, prolonged blood circulation time in animal models, and improved capability of overcoming multidrug resistance (MDR).26−30 For these reasons, we aimed at synthesizing a new kind of nitrate-functionalized TPGS derivative (TNO3), capable of self-

1. INTRODUCTION Nitric oxide (NO) is a type of low-molecular-weight endogenous signaling molecule which once got the title “Molecule of the Year” for its important effects in a number of diseases.1−3 Over the past decades, NO has shown great potential in inhibiting carcinogenesis and tumor growth.4,5 Moreover, codelivery of NO with chemotherapeutic drugs can enhance the suppression on the growth of tumor.6,7 Unfortunately, the delivery of gaseous NO to tumor directly is not really effective due to its short half-life (1−5 s) and chemical instability.8 It may undergo chemical reactions with a variety of atoms and radicals such as O2 and superoxide anion (O2•−) to generate nitrogen dioxide (NO2)9 and peroxynitrite (ONOO−),10 respectively. It can also interact with oxyhemoglobin to form methemoglobin and nitrate.11 To solve these, a number of NO donors have been developed to generate NO in situ, such as nitroprusside,12 S-nitrosothiols (RSNOs),13 N-diazeniumdiolates (NONOates),14 and the wellknown organic nitrate, nitroglycerine (GTN).15 However, most of them are low-molecular-weight compounds which may increase the exposure to reticuloendothelial system (RES) and accelerate the clearance by the body.16 In a bid to achieve extended circulation time and improve the pharmacokinetics, macromolecular NO donors have been studied in recent years, such as dendrimers, liposomes, and nanoparticles.17−19 Duong © XXXX American Chemical Society

Received: April 24, 2014 Revised: September 9, 2014 Accepted: September 15, 2014

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Scheme 1. Synthetic Scheme of TNO3 Polymer

were purchased from the laboratory animal center of Huazhong University of Science and Technology (Certificate No. SCXK 2010-0009, Wuhan, People’s Republic of China). They were kept at a temperature of 25 ± 1 °C, relative humidity of 50− 60% and in 12 h light−dark cycles. All the animals were fasted for 12 h before experiments. All animal tests were performed in accordance with the institutional ethics committee regulations and guidelines on animal welfare. 2.2. Synthesis and Characterization of TNO3. TNO3 was synthesized by a two-step method (Scheme 1). TPGS was first modified with 4-BrC. Briefly, 3.00 g of predried TPGS (1.95 mmol) and 300 μL of pyridine (3.90 mmol) were dissolved in 20 mL of anhydrous dichloromethane. 0.72 g of 4BrC (3.90 mmol) was then added into the solution dropwise in an ice bath. The mixture was then stirred at 0 °C for 4 h and further reacted at room temperature under N2 atmosphere for 20 h. The resulting solution was then precipitated in cold diethyl ether and washed twice to remove unreacted 4-BrC. After solvent evaporation, the synthesized TPGS-4-bromobutyl (TBr) was dialyzed against 1:1 (v/v) water:methanol for 72 h before freeze-drying. TBr and AgNO3 (molar ratio = 1:5) were then codissolved in 15 mL of anhydrous acetonitrile, and the mixture was stirred for 24 h. The solution was precipitated in cold diethyl ether and centrifuged at 6000 rpm for 10 min followed by dialysis against 1:1 (v/v) water:acetonitrile for 24 h. After freeze-drying, the resulting product was collected and stored at 4 °C for further use. The structures of TBr and TNO3 polymer were confirmed by 1 H NMR (Bruker AM-400 spectrometer, Switzerland) and Fourier transform infrared spectroscopy (FTIR, Bruker VERTEX 70 spectrophotometer, Germany). The conjugation of nitrate was also analyzed by ultraviolet spectrophotometer (UV, UV-1750, Shimadzu). The degree of substitution (DS) was assessed by elemental analysis (vario MICRO cube, Elementar, Germany) and calculated approximately as eq 1.

assembling into micelles with enhanced permeability and retention (EPR) effect in tumors. In vitro NO release kinetics of TNO3 as well as the cytotoxicity and apoptosis-inducing effect was further evaluated. In order to clarify the enhancement of antitumor activity of chemotherapeutic drugs by codelivery of TNO3, doxorubicin (DOX) was selected as the model drug. The cellular uptake, cell cytotoxicity, and apoptosis of DOX with TNO3 (DOX&TNO3) toward hepatocarcinoma cell line (HepG2) were investigated. Pharmacokinetics as well as tumor tissue distribution assays were performed to study the behavior of DOX&TNO3 in vivo. Antitumor activity of DOX&TNO3 in vivo was further evaluated on murine hepatic carcinoma H22 and sarcoma S180 tumor-bearing mice.

2. MATERIALS AND METHODS 2.1. Materials. TPGS, 4-bromobutyl chloride (4-BrC, 95%), silver nitrate (AgNO3, 99.8%), trypsin-EDTA, and glutathione (GSH, 98%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Doxorubicin hydrochloride was obtained from Beijing Huafeng United Technology Co., China. Nitroglycerine injection (5 mg/mL) was obtained from Shanxi Kangbao Biological Product Co. RPMI-1640, and DMEM medium was purchased from Gibco BRL (Gaithersberg, MD, USA). Penicillin−streptomycin and fetal bovine serum (FBS) were acquired from Hyclone (USA). MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Hoechst 33342 were purchased from Biosharp, South Korea. The solvents of analytical grade were obtained from Sinopharm, China. Radioimmunoprecipitation assay (RIPA) buffer, BCA Protein Quantitation Kit, Griess reagent, and 3amino-4-aminomethyl-2′,7′-difluorescein, diacetate (DAF-FM DA) probe were obtained from Beyotime Institute of Biotechnology, China. Polyvinylidene difluoride (PVDF) membrane was purchased from Bio-Rad, USA. Antibodies against cleaved Caspase-3 were purchased from Cell Signaling Technology, USA. SuperSignal West Pico chemiluminescent substrate was obtained from Thermo Scientific, USA. The human hepatocarcinoma cell line (HepG2) was acquired from the American Type Culture Collection (ATCC, USA). Murine hepatic carcinoma cell line (H22) and sarcoma cell line (S180) were purchased from Shanghai Institute of Cell Biology (Shanghai, China). Female Sprague−Dawley (SD) rats of weight 200 ± 20 g and Kunming (KM) mice of weight 20 ± 2 g

DS (%) = [(%age of N element in TNO3) − (%age of N element in TPGS)] /[(theor %age of N element in TNO3) − (%age of N element in TPGS)] × 100% B

(1)

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were subsequently rinsed twice with PBS to remove excess probe. TNO3 at a concentration of 15 μM was added to the cells, which were pretreated with DAF-FM DA. After 4 h incubation, the cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Following fixation, cells were washed three times with PBS and stained with Hoechst 33342 for 8 min. The cells were further rinsed with PBS and mounted on slides for observation by confocal laser scanning microscopy (Leica TCSNT1, Germany). The fluorescence produced by the probe reacting with NO was measured as above. 2.4.4. Intracellular Release of NO Measured by Flow Cytometry. Briefly, HepG2 cells were seeded into 6-well plates at a density of 104 cells/well and cultured at 37 °C for 24 h prior to the experiment. The cells were then washed twice with PBS and cultured in RPMI 1640 medium containing 5 μM DAF-FM DA at 37 °C for 40 min. TNO3 micelles and GTN were then added with RPMI 1640 medium at NO concentration of 10 μM. Untreated cells were used as control. After incubation at 37 °C for 4 h, the cells were trypsinized, collected, and then dispersed in 300 μL of PBS. The stained cells were analyzed using a flow cytometry (Becton Dickinson, San Jose, CA). 2.5. Intracellular Uptake of DOX by Confocal Microscopy and Flow Cytometry. Confocal microscopy was used to compare the cellular uptake of DOX and DOX&TNO3 by HepG2 cells. The cells were seeded in a 24well plate. After the cells reached 70−80% confluence, the medium was substituted by RPMI 1640 medium containing 5 μM DAF-FM DA at 37 °C for 40 min. Afterward, the cells were incubated with either a mixture of 5 μg/mL DOX and 20 μg/ mL TNO3 or 5 μg/mL DOX alone for 2 h at 37 °C. The wells were carefully washed twice with cold PBS to remove the excess drug not taken up by the cells. After fixation with 4% paraformaldehyde for 15 min, the cells were washed twice with cold PBS. The cell nuclei were then stained with Hoechst 33342 for 8 min and rinsed with PBS to remove the free dye. The cells were viewed and imaged by confocal microscopy. Quantitative cellular uptake of DOX was determined by flow cytometry. HepG2 cells were seeded into 6-well plates at a density of 104 cells/well and cultured at 37 °C for 24 h prior to the experiment. Then, a mixture of 5 μg/mL DOX and 20 μg/ mL TNO3 or 5 μg/mL DOX alone was added. After incubation at 37 °C for 2 and 4 h, the cells were trypsinized, collected, and dispersed in 300 μL of PBS. 2.6. In Vitro Cell Cytotoxicity and Apoptosis Assay. 2.6.1. MTT Assay. For cytotoxicity assays, a comparison between TNO3 and GTN, as well as DOX and DOX&TNO3, was investigated on HepG2 cells. The cells were cultured in RPMI 1640 medium with 5% CO2 at 37 °C and supplemented with 10% fetal bovine serum, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin. HepG2 cells (5000 cells/well) were seeded in 96-well plates and incubated with 100 μL of culture medium overnight at 37 °C. The medium was substituted for medium containing samples at serial concentrations of 0.01, 0.1, 1, 5, and 10 μM NO (GTN and TNO3) or 0.01, 0.1, 1, 5, and 10 μg of DOX/mL (DOX and DOX&TNO3). TNO3 of DOX&TNO3 was in serial concentrations of 0.01, 0.1, 1, 5, and 10 μM NO. The viability of HepG2 cells was determined using MTT assay at designed time intervals (24, 48, and 72 h). The plate was incubated at 37 °C for 4 h after 10 μL of MTT had been added into each well. The precipitant was then dissolved by 150 μL of DMSO using an automated shaker. The

H NMR data of TPGS, TBr, and TNO3 (CDCl3, ppm): 0.86 (a, 12H, −CH(CH3)CH3 and −CH2CH(CH3)CH2−, TPGS), 1.00−1.80 (b, −CH2CH2CH2CH(CH3)CH2CH2CH2CH(CH 3 )CH 2 CH 2 CH 2 CH(CH 3 )CH 3 and −OC(CH 3 )(CH2)−, TPGS), 1.89−2.09 (c, 9H, CH3Ph, TPGS), 2.59 (d, 2H, PhCH2CH2−, TPGS), 2.80 (e, 2H, −PhOCOCH2CH2COO−, TPGS), 2.95 (f, 2H, −PhOCOCH2CH2COO−, TPGS), 4.25 (g, 4H, −COOCH2CH2O−, TPGS), 3.65 (h, 92H, −OCH2CH2O−, TPGS), 2.18 (j, 2H, −CH2−CH2− CH2−, TBr), 2.54 (k, 2H, −CH2−Br, TBr), 3.46 (i, 2H, −CO−CH2−CH2−, TBr), 2.49 (l, 2H, −COCH2−, TNO3), 4.50 (n, 2H, −CH2−O−NO2, TNO3), 2.10 (m, 2H, −CH2− CH2−CH2−, TNO3). 2.3. Preparation and Characterization of TNO 3 Micelles. TNO3 micelles were prepared by thin film hydration method. Twenty milligrams of TNO3 was dissolved in chloroform, and then the solvent was removed by rotary evaporation. The formed film was dried in vacuum for 1 h prior to hydration with 5 mL of phosphate-buffered saline (PBS, pH 7.4). The sample was further incubated at 37 °C for 30 min followed by sonication for 5 min. The resultant micelles were filtered through 0.22 μm poly(ether sulfone) syringe filter. Particle size of TNO3 micelles was measured by dynamic light scattering (DLS, Zeta Plus, Brookhaven Instruments, USA). Surface morphology was observed by transmission electron microscopy (TEM, Tecnai G2 20, FEI, The Netherlands). TPGS micelles were prepared in a similar way as the above description. The stability of TPGS and TNO3 micelles in PBS at different pH with/without GSH was monitored by DLS. 2.4. In Vitro NO Release of TNO3. 2.4.1. NO Release in PBS. Kinetics of NO release of TNO3 in PBS was monitored at 37 °C with comparison to GTN. Briefly, TNO3 micelles or GTN which contained 200 μM NO in PBS (pH 7.4) with the presence or absence of 10 mM GSH were placed in a water bath shaker at 120 rpm. Fifty microliters of the medium was collected and replaced with equal volumes of fresh media at predetermined intervals. The concentration of nitrite, the stable breakdown product of gaseous NO, was evaluated using the Griess assay as an indirect measurement of NO concentration in PBS medium. The assay was based on the reaction between sulfanilamide, N-1-naphthylethylenediamine dihydrochloride, and nitrite, under acidic conditions to produce a compound which can be measured by UV at 540 nm.31 Experiments for all samples were performed thrice. 2.4.2. NO Release in Cells. To determine NO release profiles of TNO3 in cells, HepG2 cells were seeded in a 96-well plate. After incubation for 24 h, cells were treated with TNO3 or GTN which contained 8 μM NO with 100 μL of phenol red free DMEM medium. At 1, 2, 4, 8, 12, 24, 48, 72, and 96 h, 50 μL of medium was taken from three wells and mixed with 50 μL of Griess reagent to determine the concentration of nitrite. The absorbance of the solution was measured at 540 nm by using a microplate reader (Multiskan MK3, Thermo, USA). 2.4.3. Intracellular Release and Distribution of NO by Confocal Microscopy. HepG2 cells (5000 cells/well) were seeded in a 24-well plate and incubated for 24 h. The medium was then replaced by RPMI 1640 medium containing 5 μM DAF-FM DA and further incubated at 37 °C for 40 min.32 DAF-FM DA is cell-permeant and can passively diffuse across the cellular membrane. Once in cells, it is deacetylated by intracellular esterases to become 3-amino-4-aminomethyl-2′,7′difluorescein (DAF-FM). After reacting with NO, DAF-FM can be detected at 470/585 nm (excitation/emission). The cells C

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and stored at −20 °C until analysis. The concentration of DOX in plasma was analyzed by high-performance liquid chromatography (HPLC). Briefly, the serum was extracted by chloroform/methanol (10:1, v/v) and vortexed for 5 min. The mixture was centrifuged at 11,000 rpm for 10 min, and the lower solution was collected and dried by nitrogen. The residue was redissolved in methanol, and the solution was centrifuged at 11,000 rpm for 10 min. Twenty microliters of the supernatant was used for the analysis. The standard curve was linear and ranged from 0.05 to 50 μg/mL (R2 = 0.9994). HPLC analysis was performed by using a reverse phase column (Restek C18 5 μm, 150 mm × 4.6 mm). Acetonitrile and 0.067 mM KH2PO4 buffer (25:75, v/v) whose pH was adjusted to 4.21 with H3PO4 were used as the mobile phase with a flow rate of 1.0 mL/min. The fluorescence detector was operated at 470 nm (excitation) and 585 nm (emission). The pharmacokinetic parameters were calculated using the drug and statistics (DAS) software (version 2.1.1; Mathematical Pharmacology Professional Committee, Shanghai, China). 2.8. Tumor Tissue Distribution Assay. Tumor-bearing mice were prepared by inoculating H22 cells (1 × 107) in the right flank of female KM mice (5−7 weeks old), and the tumor was allowed to grow for 3 days. The mice were divided into 2 groups (n = 8) when the tumor volume reached ∼500 mm3. The mice in one group were given free DOX, and the other group was DOX&TNO3 (20 mg of TNO3/kg) with a single injection of 5 mg of DOX/kg via the tail vein. The mice were then sacrificed by cervical dislocation at 1, 2, 4, 8, 24, 48 h, and the tumor was immediately excised. Tumor tissues were then lightly washed and blotted to remove any excess blood. The tissues were homogenized and extracted with chloroform:methanol (10:1, v/v), and the extracts were then subjected to HPLC assay according to the method of pharmacokinetic study. 2.9. Tumor Treatment. The animal model used was H22/ S180-transplanted solid tumor bearing mice (KM mice, 5−7 weeks old, 20 ± 2 g). The mice were subcutaneously injected at the lower right axilla with 0.2 mL of H22/S180 cell suspension containing 107 cells. After inoculation, the tumor volume was daily measured by digital caliper and calculated as length × width2/2. Upon the growth of tumor volume reaching 20−100 mm3, the mice were randomly distributed into six groups (n = 8). After iv injection through the tail vein with saline, GTN, TPGS, TNO3, DOX, and DOX&TNO3 (20 mg TNO3/kg) at a dose of 5 mg of DOX/kg on day 1, 3, 5, and 7, the tumor size was measured every day to evaluate the antitumor efficiency. At the end of the experiment, H22 tumor tissues were collected and weighed. Tissues were then fixed in 4% paraformaldehyde for 3 days, embedded in paraffin, serially sectioned, and stained with hematoxylin eosin as per the standard protocol. The permeation effect of TNO3 was verified by comparing DOX concentration in the central 2 mm3 part of tumor tissues by frozen section method. 2.10. Statistical Analysis. Every experiment was repeated at least three times. All results were reported as the mean ± standard deviation (SD). The differences of the mean were calculated by one-way ANOVA using SPSS software (version 19.0). The statistical significance level was set as a probability of P < 0.05.

absorbance of each well was read at 570 nm by a microplate reader. All the experiments were done with seven parallel samples. Relative cell viability was calculated as a percentage in relation to untreated control cells. IC50 (concentration resulting in 50% inhibition of cell growth) value of the drugs was determined by SPSS software (version 19.0). The experiment was performed three times. Coefficient of drug interaction (CDI), a synergistic factor, was calculated as eq 2.33 CDI value less than, equal to, or larger than 1.0 indicates that the drugs are synergistic, additive, or antagonistic, respectively. CDI less than 0.7 indicates significantly synergistic effect between combinational drugs. CDI =

IC50(DOX&TNO3) IC50(DOX) × IC50(TNO3)

(2)

2.6.2. Intracellular ATP Level Assay. HepG2 cells were seeded into 12-well plates. After the cells reached 90% confluence, they were then treated with GTN or TNO3 at the same concentration of 10 μM NO, respectively. The other groups were separately incubated with DOX and DOX&TNO3 at 5 μg of DOX/mL with 20 μg of TNO3/mL. Intracellular ATP levels were determined by the luciferrin−luciferase-based ATP luminescence assay kit (Beyotime Institute of Biotechnology, China) as instructed by protocol. 2.6.3. Hoechst 33342 Staining. The cells were seeded into a 24-well plate (104 cells per well) and incubated for 24 h. They were then treated with GTN or TNO3 at the same concentration of 10 μM NO, respectively. The other groups were separately incubated with DOX and DOX&TNO3 at 5 μg of DOX/mL with 20 μg of TNO3/mL. After incubation for another 24 h, the cells were rinsed three times with cold PBS and then fixed with 200 μL of 4% paraformaldehyde for 15 min. The cells were further washed three times with 500 μL of PBS for 3 min and stained with 200 μL of Hoechst 33342 for 8 min. The cells were then mounted on a glass slide for observation by fluorescence microscopy (IX71, Olympus, Tokyo, Japan). 2.6.4. Western Blot for Caspase-3 Activation. HepG2 cells were seeded into 6-well plates. After the cells reached 90% confluence, they were treated with GTN, TNO3, DOX, and DOX&TNO3 at the same concentration of the experiment in 2.6.2. After incubation for 24 h, cells were lysed in RIPA lysate buffer containing 1% Triton X-100, 1% deoxycholate, and 0.1% SDS and the protein content in lysate was analyzed by BCA protein assay kit. 40−60 μg of protein/sample was loaded on SDS−polyacrylamide gels (SDS−PAGE) and transferred to PVDF membrane which was blocked with 5% nonfat milk. The membrane was then incubated with antibodies detecting Caspase-3 overnight at 4 °C, with β-actin used as the control. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG were applied for 1 h at room temperature. SuperSignal West Pico chemiluminescent substrate was used to detect chemiluminescence, and blots were imaged using GeneGnome5 gel imaging and analysis systems (Synoptics Ltd., U.K.). Densitometric analyses of protein abundance were determined by ImageJ software. 2.7. Pharmacokinetic Study. Female SD rats which weighed 200 ± 20 g were divided into two groups (n = 3). The rats were injected intravenously (iv) with DOX and DOX&TNO3 respectively (40 mg of TNO3/kg) at a dose of 10 mg of DOX/kg. About 300 μL of blood was taken from the tail vein at 0.5, 1, 2, 4, 8, 12, 24, and 72 h. After centrifugation at 3500 rpm for 10 min, 100 μL of the supernatant was collected

3. RESULTS AND DISCUSSION 3.1. Characterization of TNO3. The chemical structure of TBr and TNO3 was characterized by 1H NMR (Figure S1A in D

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According to the DLS analysis, the hydrodynamic diameter of TNO3 micelles was 133.9 ± 0.9 nm, which was much larger than that of TPGS micelles (∼18 nm). The size change of TNO3 micelles may be attributed to the conjugation of −CH2CH2CH2ONO2 increasing the hydrophobic content of the whole system and the hydrophobic−hydrophilic−hydrophobic triblock structure of TPGS-ONO2 limiting the free volume of the PEG segment. We further measured the size changes of TNO3 and TPGS micelles at various designated pH values ranging from 7.4 to 3.5 (Figure S2 in the Supporting Information). Compared to the unchanged property of TPGS micelles, the size of TNO3 micelles exhibited a slight increase from 133.9 ± 0.9 nm to 148.7 ± 1.0 nm after the pH was adjusted to a value below 5.5. Moreover, a structure of spherical shape was observed in TEM images with comparable particle size to the ones measured by DLS. The stability of micelles in PBS at different pH with/without GSH was shown as Figure 1B. The micelles of TNO3 remained stable in PBS (pH 7.4 and 6.8). However, the diameter of micelles in PBS (pH 5.5) was increased from 132.6 ± 0.7 nm to 159.4 ± 3.0 nm in 8 h. This may be related to the protonation of the NO3 group under acidic condition. A large number of protons were accumulated into micelles that increased charge repulsion. As a result, the size of micelles would be enlarged.37,38 Moreover, when micelles were formed in PBS containing 10 mM GSH, the size of micelles was decreased to 113.1 ± 0.4 nm and kept for 96 h. This may be related to the ionic strength in PBS with the high concentration of GSH and the stabilization effect of TPGS. 3.3. In Vitro NO Release of TNO3 Micelles. The release kinetics of NO from TNO3 micelles and GTN was evaluated by Griess reagent in PBS (pH 7.4) and PBS containing 10 mM GSH at 37 °C, respectively (Figure 2A). GTN and TNO3 only released around 10% (15 μM) NO in 144 h while about 90% (140 μM) NO was released in PBS with 10 mM GSH. It seemed that the behavior of NO release from the donors may be redox sensitive. However, when TNO3 was incubated with GSH for 12 and 24 h, only 9.4 ± 0.1% (14.1 ± 0.2 μM) and 46.5 ± 2.1% (69.8 ± 3.2 μM) of NO content were cumulatively released compared with 63.1 ± 0.1% (94.7 ± 0.2 μM) and 80.1 ± 1.1% (120.2 ± 1.7 μM) NO released by GTN, respectively. This may be related to the high molecular weight of TNO3 and its micelle structure which could delay the reaction with GSH. To further verify the phenomena, NO release property of TNO3 was investigated in DMEM medium with HepG2 cells. As shown in Figure 2B, after incubation with cells for 96 h, 90.5 ± 2.0% (7.2 ± 0.2 μM) NO in TNO3 was released while only 68.0 ± 1.4% (5.4 ± 0.1 μM) was released in GTN, which were much higher than that generated by TNO3 in medium (16.6 ± 0.2%, 1.3 ± 0.1 μM), GTN in medium (12.2 ± 0.9%, 1.0 ± 0.1 μM), and HepG2 cells (5.0 ± 0.5%, 0.4 ± 0.1 μM). Intracellular NO release was detected by observing the fluorescence reaction with DAF-FM DA by confocal microscopy (Figure 2C). The presence of green fluorescence in the control was attributed to the reaction of DAF-FM with NO endogenously produced by the cells. In contrast, significantly higher fluorescence of TNO3 group was detected in the cytoplasm. Flow cytometry analysis (Figure 2D) also demonstrated that TNO3 generated much more NO in cells with ∼3-fold higher fluorescence intensity than the control. On the other hand, GTN, a small molecule NO donor, exhibited very dim green light, which may be an indication of the lower NO release and limited cell uptake.

the Supporting Information) and FTIR spectrum (Figure S1B in the Supporting Information). In the 1H NMR spectrum of TBr, the peaks at 2.18 and 2.54 ppm which were assigned to the −CH2− and −CH2−Br, respectively, showed that 4-BrC was conjugated with TPGS. The newly appearing signals at 2.49 and 4.50 ppm in the TNO3 spectrum which belonged to the −COCH2− and −CH2−O−NO2, respectively, verified the formation of TPGS-nitrate.34 The structure of TNO3 was further studied by FTIR. The organic nitrate peaks come out at 1286 cm−1 (NO2 symmetric stretch) and 1634 cm−1 (NO2 asymmetric stretch), indicating the successful synthesis of TNO3.35 This result was also confirmed by the UV spectrum of TPGS, TBr, and TNO3 (Figure S1C in the Supporting Information). Compared to the absorption peak at 286 nm for TPGS and TBr, a new peak was exhibited at 256 nm for TNO3, which may be attributed to the conjugation of nitrate.36 The nitrate content of TNO3 was evaluated by elemental analysis (Figure S1D in the Supporting Information) with DS of 54.8 ± 1.7%. 3.2. Characterization of TNO3 Micelles. The TNO3 micelles were characterized by DLS and TEM (Figure 1A).

Figure 1. Transmission electron microscopy of TNO3 micelles (A) and stability of TNO3 micelles in PBS at different pH values with/ without GSH (B). E

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Figure 2. In vitro NO release of TNO3 and GTN incubated in PBS at different pH values with/without 10 mM GSH(A), and with HepG2 cells in phenol red free medium (B) by a Griess reagent. Intracellular NO release of TNO3 by confocal laser scanning microscopy (C) and flow cytometer (D) compared to GTN.

necrosis than GTN. The low levels of NO (500 nM) released by TNO3 may promote cancer cell cytotoxicity and apoptosis.5,8,42−44 Moreover, NO donors with higher NO release capability may be more potent in inhibition of DNA synthesis and mitotic activity in the S-phase compared to lower NO release donors.45 Besides the direct inhibition on tumor growth, NO has been found to improve the efficiency of DOX via a NO-dependent mechanism.46 3.5. Enhanced Intracellular Uptake of DOX by TNO3. To investigate the synergistic effect of TNO3 on DOX, the cellular uptake of DOX and DOX&TNO3 was first analyzed on HepG2 cells by flow cytometry measurement and confocal microscopy. As shown by the results of flow cytometry (Figures 4A and 4B), DOX&TNO3 exhibited a significantly enhanced cellular uptake of DOX. The mean fluorescent intensity (MFI) was 2.52-fold (991) and 3.42-fold (1941) higher than that of free DOX after incubation for 2 and 4 h, respectively. Confocal microscopy images (Figure 4C) further indicated the enhanced cellular uptake of DOX by TNO3. A higher concentration of DOX was observed in the nuclei as compared with free DOX when codelivering with TNO3 for 2 h. Meanwhile, a significantly high NO release by TNO3 was detected throughout the cytoplasm, which may be the reason for the enhanced DOX uptake of cancer cells.47

As mentioned above, TNO3 micelles exhibited a significant stability with minimum NO leakage in PBS but a sustained and much higher NO release in cancer cells with the help of GSH. The glutathione S-transferase (GST), relatively acidic environment, and quantity of free radical may also promote NO releasing ability.16,39 As previously reported, there was a 5000fold higher concentration of GST and GSH in tumor cells compared with the extracellular environment.40,41 These may explain why TNO3 micelles exhibited a much higher NO release in tumor cells as compared with that in free medium. 3.4. In Vitro Antitumor Activity of TNO3. The antitumor activity of TNO3 was first measured and compared with GTN by MTT assay on HepG2 cancer cells (Figure 3A). Cell viability of GTN at all concentrations was beyond 100%, suggesting no obvious cytotoxicity. In contrast, the cytotoxicity of TNO3 was obvious in a concentration-dependent tendency and the IC50 values were 45.86 ± 6.61, 27.47 ± 5.03, and 14.38 ± 3.41 μg/mL at 24, 48, and 72 h (Table 1), respectively. The apoptosis assay was then conducted by Hoechst staining of nuclei (Figure 3B). Compared to GTN, much more typical apoptotic features of chromatin condensation and apoptotic body formation were observed in the cells treated with TNO3. The intracellular ATP level assay was further conducted to confirm this tendency (Figure 3C). The ATP level of the cells treated with GTN was 79.3 ± 5.9% compared to the control while a significant reduction to 41.1 ± 4.6% was observed on the TNO3 group. As seen from these values, TNO3 may exhibit a better activity on cytotoxicity and inducing apoptosis or F

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Figure 3. In vitro cytotoxicity of GTN, TNO3, DOX, and DOX&TNO3 against HepG2 cells after being treated for 24, 48, and 72 h, respectively (A). Nucleus apoptosis assay of HepG2 cells treated with control (a), GTN (b), TNO3 (c), DOX (d), and DOX&TNO3 (e) respectively for 24 h (B). Intracellular ATP levels (C) and the expressions of cleaved Caspase-3 protein with the ratio of Caspase-3/β-actin (D) in HepG2 cells treated with control, GTN, TNO3, DOX, and DOX&TNO3 respectively after 24 h incubation. *: p < 0.05. **: p < 0.01. ***: p > 0.05.

were 6.25-, 1.42-, and 1.48-fold lower than those of free DOX at 24, 48, and 72 h, respectively (Table 1). This suggested the significant cytotoxicity enhancement effects of TNO3 in the treatment with DOX. In order to further evaluate the interaction between DOX and TNO3, CDI was calculated. CDI values of DOX&TNO3 were 0.004, 0.026, and 0.046 for 24, 48, and 72 h, respectively, which were much lower than the synergistic value of 0.7, indicating a significant synergistic effect of TNO3 with DOX. This may be related to the high quantity of NO released in cancer cells, in which way it may not only induce cytotoxicity but also chemosensitize tumor cells to chemotherapeutic drugs.48 Moreover, the investigations of Hoechst staining of nuclei, intracellular ATP level assay, and Western blot for Caspase-3 activation were conducted on HepG2 cells. Compared to free DOX, significant split of nuclei was observed with DOX&TNO3 (Figure 3B). In addition, an obvious decrease in ATP level to 9.9 ± 6.8% was observed, which was 3.2-fold lower than that of free DOX (31.8 ± 0.4%) (Figure 3C). In Western blot assay

Table 1. IC50 of GTN, TNO3, DOX, and DOX&TNO3 on HepG2 Cells after Different Incubation Time (n = 6) IC50 a

GTN TNO3a DOXb DOX&TNO3b a

24 h

48 h

72 h

>100 45.86 ± 6.61 16.54 ± 0.59 2.65 ± 0.03

>100 27.47 ± 5.03 0.69 ± 0.06 0.48 ± 0.05

>100 14.38 ± 3.41 0.59 ± 0.04 0.39 ± 0.02

μg/mL. bμg of DOX/mL.

3.6. Enhanced Cell Cytotoxicity and Apoptosis of DOX by TNO3. To evaluate the combinational effect of TNO3 on DOX, the cytotoxicity of DOX&TNO3 on HepG2 cells was assessed as a comparison to free DOX (Figure 3A). The cell viabilities were 60% and 20% after exposure to free DOX of 10 μg/mL for 24 and 72 h, respectively. However, DOX&TNO3 exhibited a significantly low cell viability of 10% after incubation for 24 h. Moreover, IC50 values of DOX&TNO3 were 2.65 ± 0.03, 0.48 ± 0.05, and 0.39 ± 0.02 μg/mL, which G

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Figure 5. Pharmacokinetics behavior after iv injection of DOX or DOX&TNO3 at a dose of 10 mg of DOX/kg to SD rats (n = 3) (A). Drug levels in tumor tissue after iv injection of DOX and DOX&TNO3 into KM tumor-bearing mice at a dose of 5 mg of DOX/kg (n = 3) (B). Results are given as mean ± SD. *: p < 0.05. **: p < 0.01. ***: p > 0.05.

Table 2. Pharmacokinetic Parameters in Rats after Iv Injection of DOX or DOX&TNO3 at a Dose of 10 mg of DOX/kg and 40 mg of TNO3/kga Figure 4. Intracellular uptake of DOX and DOX&TNO3 by flow cytometry measurement (A) with MFI (B) and confocal microscopy images (C).

(Figure 3D), more protein Caspase-3 (17 kDa) was presented in DOX&TNO3 and the ratio of the Caspase-3/β-actin (0.39 ± 0.06) was much higher than the values for control (0.08 ± 0.01), GTN (0.12 ± 0.01), TNO3 (0.18 ± 0.02), and DOX (0.25 ± 0.05). These also confirmed the TNO3-induced enhancement on cell cytotoxicity of DOX.6,46,49 3.7. Pharmacokinetic Study of DOX&TNO 3. To investigate the effect of TNO3 on DOX pharmacokinetics, DOX&TNO3 and free DOX were administered to SD rats, respectively. The plasma DOX concentration−time profiles are shown in Figure 5A, and the corresponding pharmacokinetic parameters are presented in Table 2. It can be seen that, compared with free DOX, DOX&TNO3 showed an 8.63- and 2-fold higher DOX concentration after 0.5 and 48 h administration, respectively. Interestingly, DOX&TNO3 exhibited a significantly longer retention time in the blood as opposed to free DOX. DOX&TNO3 demonstrated 13.7-fold area under the curve (AUC(0−72)), 6.4-fold mean residence

a

params

unit

DOX

DOX&TNO3

AUC(0−t) AUC(0−∞) MRT(0−t) MRT(0−∞) t1/2 Tmax CL V Cmax

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

0.58 ± 0.28 0.66 ± 0.36 2.08 ± 0.49 3.11 ± 1.44 2.62 ± 2.01 0.5 20.12 ± 13.83 65.04 ± 42.27 0.27 ± 0.15

8.24 ± 4.44 9.52 ± 4.40 13.45 ± 4.69 28.80 ± 8.58 38.44 ± 18.23 0.5 1.05 ± 0.80 58.24 ± 10.92 3.02 ± 1.27

The values are shown as mean ± SD (n = 3).

time (MRT), 14.7-fold half-life (t1/2), and substantially lower values of clearance (5.4%) compared to those of free DOX. This study suggested that TNO3 prolonged the circulation time of DOX in vivo. 3.8. Tumor Tissue Distribution of DOX&TNO3. We further carried out the tumor tissue distribution assay to study the DOX retention in H22-tumor bearing mice after treatment with DOX&TNO3 and free DOX, respectively (Figure 5B). The DOX concentrations of free DOX were 3.1 ± 0.5 and 2.3 ± 0.3 μg/g tumor after 1 and 4 h administration, respectively. H

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DOX exhibited a negligible tumor growth inhibition, which might have resulted from its short circulation time and poor permeability into tumors.56 TNO3 exhibited similar tumor growth inhibition with free DOX until day 6, but it turned to be worse after day 7. Impressively, DOX&TNO3 exhibited the best antitumor activity, and the tumor volume had reduced to only 44.2% of that treated with free DOX at day 9, while the body weight of DOX&TNO3 treated mice exhibited similar behavior with that of the mice treated with free DOX (Figure 6B). After treatment, tumor tissues were collected and weighed (Figures 6C and 6D). Tumors of saline, GTN, TPGS, TNO3, and DOX groups were 2.4 ± 0.9 g, 2.2 ± 0.9 g, 2.5 ± 1.0 g, 1.4 ± 0.7 g, and 0.9 ± 0.5 g, respectively. However, the DOX&TNO3 group showed a significant reduction of tumor weight (0.5 ± 0.1 g), which was 49.5% of the group treated with free DOX. These results indicated the synergistic effect between TNO3 and free DOX on tumor-growth inhibition. This tendency was also demonstrated on S180 tumor-bearing mice (Figure S3 in the Supporting Information). DOX&TNO3 also exhibited the best antitumor activity, and the tumor weight was only 0.4 ± 0.1 g, which was 78.3% of that treated with free DOX. The inhibition results were not as good as for the H22 tumor model. This may be attributed to the fact that DOX is not particularly efficient in treating sarcoma S180 tumors. H&E staining of the tumor section is presented in Figure 7. Compared with TNO3 or free DOX, cell nuclei apoptosis and

DOX&TNO3 produced increased accumulation of DOX in the tumors compared to free DOX, and resulted in DOX concentration of 4.2 ± 0.1 and 3.2 ± 0.1 μg/g tumor at 1 and 4 h, respectively. It was noteworthy that the mice treated with DOX&TNO3 demonstrated about 1.4-fold higher DOX levels in tumors for the first 4 h, but showed similar levels after 8 h compared to that of free DOX. This may have resulted from the effects of NO on the tumor blood vessels and tumor tissues. As reported previously, tumor blood vessels were extremely irregular and tortuous and had incomplete endothelial linings and basement membrane.50 As a result, blood flow was often highly irregular, and the vessels were much leakier than those in normal tissues.51 Moreover, as compared with normal tissues, solid tumors may have fewer functional lymph vessels, which contributed to the increased interstitial fluid pressure within tumor tissues.52 The increased interstitial pressure in solid tumors further decreased tumor perfusion. However, NO in vivo can develop a potent effect on the dilation of blood vessels. Therefore, when TNO3 micelles accumulated into tumors by EPR effect and constantly released NO, the interstitial pressure can be reduced by the dilation of blood vessels. 53 Consequently, DOX was able to accumulate in tumors in a short period.54 However, the levels of DOX in the tumor tissue became comparable between the free DOX and DOX&TNO3 groups after 8 h. As reported, low-molecular-weight compounds diffuse to tissues and organs against a concentration gradient until an equilibrium results. As a result, their concentrations in tumors are lower than those in blood. Furthermore, rapid excretion into the blood would make it difficult to retain them in tumors for a long time.55 Therefore, after most DOX in plasma was cleared, the levels of DOX in the tumor tissue became comparable between the free DOX and DOX&TNO3. 3.9. Enhanced Tumor Growth Inhibition of DOX by TNO3. In order to evaluate the antitumor activity of DOX&TNO3, we further explored its inhibition efficiency on H22 tumor-bearing mice with rapid and sustained tumor growth. As shown by Figure 6A, GTN and TPGS failed to inhibit the tumor growth at the end of the assay in a manner similar to that of saline. In contrast, TNO3, free DOX, and DOX&TNO3 showed different tumor suppression effects. Free

Figure 7. Images of H&E-stained tumor sections excised from subcutaneous H22 tumor-bearing mice on the ninth day after different treatments: saline (A), GTN (B), TPGS (C), TNO3 (D), DOX (E), and DOX&TNO3 (F). Images were obtained under Leica microscope using a 40× objective.

vacuoles were more severe in the DOX&TNO3 group. But these were not observed in any of the saline, GTN, and TPGS treated tumors, where the tumor cells were spindle and round with a rich cytoplasm and more nuclear division. The results were consistent with the in vitro results above. Furthermore, tumor tissues treated with DOX or DOX&TNO3 were fixed and observed by frozen section to evaluate the permeation effects of TNO3 (Figure 8). Slight red fluorescence was observed in the images of free DOX, which indicated that a small amount of DOX was accumulated in solid tumors. However, in the group of DOX&TNO3, much stronger fluorescence intensity of DOX was detected in the tumor tissues at the end of tumor treatment. This may be related to the enhanced perfusion generated by NO.53 It seems that TNO3 may not only enhance the tumor growth inhibition but also realize a deeper tumor penetration for DOX.

Figure 6. In vivo antitumor efficiency of different treatment groups in H22 tumor-bearing mice. KM mice were injected with saline, GTN, TPGS, TNO3, DOX, and DOX&TNO3 on alternate days. Tumor volume (A); relative body weight of tumor-bearing mice (B); tumor weight of tumor-bearing mice (C); images of tumor tissues (D). Data was presented as mean ± SD (n = 6). *: p < 0.05. **: p < 0.01. ***: p > 0.05. I

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ABBREVIATIONS USED NO, nitric oxide; GTN, nitroglycerine; DOX, doxorubicin; CMC, critical micelle concentration; EPR, enhanced permeability and retention; TPGS, D-α-tocopheryl polyethylene glycol succinate; TNO3, TPGS nitrate; TBr, TPGS-4bromobutyl; DAF-FM DA, 3-amino-4-aminomethyl-2′,7′-difluorescein, diacetate; MDR, multidrug resistance; AUC, area under the concentration−time curve; 4-BrC, 4-bromobutyl chloride; DS, degree of substitution; GSH, glutathione; GST, glutathione S-transferase; DMSO, dimethyl sulfoxide; CDI, coefficient of drug interaction; ATP, adenosine triphosphate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MRT, mean residence time

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Figure 8. Frozen section images of the 2 mm3 part of tumor tissue treated with DOX or DOX&TNO3.

4. CONCLUSIONS In summary, TNO3, a polymer modified with nitrate to achieve NO releasing ability, was successfully synthesized here. It may self-assemble into micelles and achieve sustained NO release in tumor cells. Besides, TNO3 exhibited a good capability of inducing cancer cell cytotoxicity and apoptosis in vitro and synergistic antitumor efficiency while combined with the anticancer drug DOX. We thus believe that TNO3 may be a potential candidate to deliver NO into tumors and realize the enhancement of chemotherapeutic effects.

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ASSOCIATED CONTENT

* Supporting Information S

1

H NMR, FTIR, UV spectra, and elemental analysis results of rough materials, size change of micelles in PBS at different pH, in vivo antitumor efficiency of different treatment groups in S180 tumor-bearing mice, and the expressions of cleaved Caspase-3 protein in HepG2 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*(Z.Z.) Tel: +86-27-83601832. E-mail: zhipingzhang@mail. hust.edu.cn. *(L.S.) Tel: +86-27-83601832. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported by the National Basic Research Program of China (973 Program, 2012CB932500), the National Natural Science Foundation of China (21204024 and 81373360), the Doctoral Fund of Ministry of Education of China (20120142120093), the Fundamental Research Funds for the Central Universities (2014TS091 and 2014QN134), Chutian Scholar Award, and 2013 Youth Scholar Award of HUST. The authors thank Otieno Ben Oketch (Tongji School of Pharmacy, Huazhong University of Science and Technology) and Professor Hudan Liu for her help in Western blot experiment. We are also grateful to the master’s students Mingxing Yin, Ruiqi Qiu, and Qi Tan for assistance in animal tests. J

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tumor metastasis targeting peptide to deliver doxorubicin-containing liposomes to highly metastatic cancer. Biomaterials 2012, 33 (33), 8451−8460.

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dx.doi.org/10.1021/mp5003009 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX