Mitochondria and Nucleus Dual Delivery System To Overcome DOX

DOX was mainly accumulated in tumor tissue after DOX/TPP–DOX@Pasp-hyd-PEG-FA was injected to tumor-bearing nude mice by tail vein. After free DOX wa...
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Mitochondria and Nucleus Dual Delivery System To Overcome DOX Resistance Han Cui,†,‡ Meng-lei Huan,†,‡ Wei-liang Ye,† Dao-zhou Liu,† Zeng-hui Teng,† Qi-Bing Mei,§ and Si-yuan Zhou*,†,§ †

Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi’an, 710032, China Key Laboratory of Gastrointestinal Pharmacology of Chinese Materia Medica of the State Administration of Traditional Chinese Medicine, Fourth Military Medical University, Xi’an, 710032, China

§

S Supporting Information *

ABSTRACT: Doxorubicin (DOX) is a broad-spectrum chemotherapy drug to treat tumors. However, severe side effects and development of DOX resistance hinder its clinical application. In order to overcome DOX resistance, DOX/TPP−DOX@Pasp-hyd-PEG-FA micelles were prepared by using newly synthesized comb-like amphiphilic material Pasp-hydPEG-FA. Drug released in vitro from micelles showed a pH-dependent manner. DOX/TPP− DOX@Pasp-hyd-PEG-FA induced more apoptosis in KB cell and MCF-7/ADR cell than DOX@Pasp-hyd-PEG-FA. Confocal laser scanning microscopy experiment indicated that DOX/TPP−DOX@Pasp-hyd-PEG-FA delivered TPP−DOX and DOX to the nucleus and mitochondria of the tumor cell simultaneously. Thus, DOX/TPP−DOX@Pasp-hyd-PEG-FA could significantly damage the mitochondrial membrane potential. DOX/TPP−DOX@Pasphyd-PEG-FA markedly shrinked the tumor volume in tumor-bearing nude mice grafted with MCF-7/ADR cell as compared with the same dose of free DOX. DOX was mainly accumulated in tumor tissue after DOX/TPP−DOX@Pasp-hyd-PEG-FA was injected to tumor-bearing nude mice by tail vein. After free DOX was injected to tumor-bearing nude mice by tail vein, DOX widely distributed through the whole body. Therefore, mitochondria and nucleus dual delivery system has potential in overcoming DOX resistance. KEYWORDS: doxorubicin, (3-carboxypropyl)triphenylphosphonium, pH-sensitive micelle, mitochondria

1. INTRODUCTION Doxorubicin (DOX) is a broad-spectrum chemotherapy drug to treat solid tumors. However, severe nonselective toxicity and development of DOX resistance hinder its clinical use.1 Once tumor cells acquire DOX resistance, DOX is pumped out from the tumor cell before it arrives at the nucleus, which leads to the inactivation of DOX.2,3 Besides having an effect on the nucleus, if DOX accumulates in mitochondria, it can increase the formation of reactive oxygen species (ROS) and damage mitochondrial respiratory chain components, then induce lipid peroxidation of mitochondrial membrane, and finally result in the shedding of cytochrome c from mitochondria, and activation of caspase cascade.4 Thus, it is an important stragety to deliver DOX to mitochondria to overcome DOX resistance.5 Fortunately, it was reported that (3-carboxypropyl)triphenylphosphonium (TPP) exhibited high binding affinity to mitochondria due to its high hydrophobicity and cationic charge.6,7 Thus, DOX can be delivered to mitochondria by conjugating DOX with TPP. TPP−DOX conjugate was synthesized by our research group. When TPP−DOX was cultured with DOX-resistant MDA-MB-231/ADR cells, TPP− DOX mainly distributed in the mitochondria of MDA-MB231/ADR cells and showed high cytotoxicity on MDA-MB231/ADR cells in vitro.8 However, TPP−DOX conjugate lacks © 2017 American Chemical Society

tumor targeting characteristics in vivo. Therefore, it is imperative to develop a new drug carrier, which not only can deliver DOX to the nucleus of tumor cells but also can deliver TPP−DOX to mitochondria of tumor cells to overcome DOX resistance. Self-assembled polymeric micelle is one of the most promising drug delivery systems for various anticancer drugs.9 It has several advantages such as enhancing drug solubility in water, prolonging blood circulation time, antidilution characteristic in blood circulation, increasing the drug stability in vitro and in vivo, and passive tumor targeting by enhanced permeability and retention effect.10 Many synthetic and natural materials were utilized to prepare polymeric micelles. PEG is a popular hydrophilic polymeric material with low systemic toxicity. It can prevent drug carriers from absorption of plasma protein and recognition by mononuclear phagocyte system (MPS). Therefore, PEG modified micelles showed a long circulation profile.11,12 In addition, poly aspartic acid (Pasp) is a biodegradable poly amino acid, which shows the potential to Received: Revised: Accepted: Published: 746

November 8, 2016 January 18, 2017 February 1, 2017 February 1, 2017 DOI: 10.1021/acs.molpharmaceut.6b01016 Mol. Pharmaceutics 2017, 14, 746−756

Article

Molecular Pharmaceutics

temperature for 5 h. Then FA-PEG-NH2 (0.9 g, 0.2 mmol) was added into the solution. After an overnight stirring at room temperature under nitrogen, the reaction mixture was diluted with water, and the mixture was dialyzed against deionized water for 3 days by using a dialysis bag (molecular weight cutoff: 1 kDa). The solution in the dialysis bag was lyophilized by using a freeze dryer to obtain FA-PEG-ABA. Pasp (50 mg, 0.01 mmol) was dissolved in 5 mL of DMSO, hydrazine hydrate (5 mL, 0.1 mmol) was added, and the mixture was stirred for 5 h at room temperature. Then different molar FA-PEG-ABA (molar ratio of Pasp to FA-PEG-ABA was 1:1, 1:4, and 1:10, respectively) was added. After reacting for 24 h at room temperature, the reaction mixture was diluted with water, and the mixture was dialyzed against deionized water for 3 days by using a dialysis bag (molecular weight cutoff: 3 kDa). The solution in the dialysis bag was lyophilized by using a freeze dryer to obtain Pasp-hyd-PEG-FA. 2.4. Preparation and Characterization of DOX/TPP− DOX@Pasp-hyd-PEG-FA. The DOX/TPP−DOX@Pasp-hydPEG-FA was prepared by dialysis method. In brief, 10.0 mg of Pasp-hyd-PEG-FA, 2.0 mg of DOX, and 2.0 mg of TPP−DOX were dissolved in 5 mL of DMSO. Then 10 mL of deionized water was add into DMSO. The above solution was stirred at room temperature for 6 h. The mixture was dialyzed against deionized water for 3 days by using a dialysis bag (molecular weight cutoff: 8 kDa). The solution in the dialysis bag was lyophilized to obtain DOX/TPP−DOX@Pasp-hyd-PEG-FA. DOX@Pasp-hyd-PEG-FA and TPP−DOX@Pasp-hyd-PEG-FA were prepared by the same method. Beckman Coulter dynamic light scattering particle analyzer (DLS, Delsa Nano C, USA) was used to detect size, polydispersity index, and zeta potential of micelles. Transmission electron microscopy (TEM, JEOL-100CXII, Japan) was used to observe the morphology of micelles.20 The standard pyrene method was used to determine the critical micelle concentration (CMC) of the micelles.21 In order to investigate the stability of micelles, the micelles were dispersed into pH 7.4 PBS medium (containing 10% bovine serum albumin), and the particle size and polydispersity index were measured at different time points. The drug loading and encapsulation efficiency were defined as previously reported.22 The content of DOX or TPP−DOX in micelles was determined by using HPLC-MS/MS method. 2.5. In Vitro Release of DOX and TPP−DOX from DOX/ TPP−DOX@Pasp-hyd-PEG-FA Micelles. 1 mg of DOX/ TPP−DOX@Pasp-hyd-PEG-FA was dissolved in release medium (5 mL PBS, pH 5.0, pH 6.5 and pH 7.4) and moved into a dialysis bag (molecular weight cutoff: 3 kDa). The dialysis bag was then immerged in the same pH release medium (40 mL) and incubated at 37 °C. 0.5 mL of incubation solution was taken out at a previously determined time point, and 0.5 mL of fresh blank release medium was supplemented into the outside solution of the dialysis bag. The released DOX and TPP−DOX was analyzed by an HPLC−MS/MS system (Quattro Premier, Waters Corp., Milford, MA, USA). The temperature of desolvation and source was 350 and 110 °C, respectively. The desolvation gas (500 L/h) and cone gas (50 L/h) was nitrogen. The collision gas was argon at a flow velocity of 0.18 mL/min. The analytical column was Sunfire C18 column (4.6 × 250 mm, 5 μm, Waters). The mobile phase consisted of 90% acetonitrile and 10% H2O (1% formic acid). The flow rate of the mobile phase was 0.3 mL/min. The sample volume was set at 20 μL. Quantitation of DOX and TPP−DOX

replace some nonbiodegradable polymers. Pasp is used as a pure homopolymer or as a part of block copolymers. In this paper, in order to obtain a satisfactory drug loading, a pH-sensitive comb-like polymer Pasp-hyd-PEG-FA was synthesized, in which folic acid (FA) was used as a tumor specific ligand to increase the selectivity of the micelle to folate receptor positive tumor cell;13,14 PEG and Pasp were connected via pHsensitive hydrazone bond. The free TPP−DOX and DOX were coencapsulated by Pasp-hyd-PEG-FA micelles (DOX/TPP− DOX@Pasp-hyd-PEG-FA) to simultaneously deliver DOX to mitochondria and nucleus of tumor cells in vivo. The pHcontrolled release, subcellular localization, biodistribution, and antitumor activity of DOX/TPP−DOX@Pasp-hyd-PEG-FA were investigated.

2. EXPERIMENTAL SECTION 2.1. Materials. Doxorubicin was bought from Hisun Pharmaceutical Co. (Zhejiang province, China). (3Carboxypropyl)triphenylphosphonium (TPP), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDCI), tert-butyl carbazate, triethylamine (TEA), 4-acetylbenzoic acid (ABA), folic acid (FA), trifluoroacetic acid (TFA), and polyaspartic acid (average molecular weight 5000 Da, Pasp) were purchased from J&K CHEMICA (Beijing, China). H2N-PEG-NH2 (average molecular weight 4000 Da) was purchased from Yare Biotech Inc. (Shanghai, China). 3-(4,5-Dimethylthiaol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), RPMI1640 medium, LysoTracker green, MitoTracker green, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) mitochondrial membrane potential detection kit, and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Invitrogen Corporation (Carlsbad, CA, USA). 2.2. Cell Lines and Culture Conditions. KB cells (human oral cavity epidermal carcinoma cell line, folate receptor overexpression),15−17 MCF-7 cells (human breast cancer cell line, folate receptor positive cell line),15−19 and DOX-resistant MCF-7/ADR cells were purchased from Institute of Biochemistry and Cell Biology (Chinese Academy of Science, Shanghai, China). MCF-7/ADR cells were cultured with DOX (2 μmol/L) for 24 h once a week to maintain the DOXresistant property. Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C and 5% CO2 under fully humidified conditions. 2.3. Synthesis of Pasp-hyd-PEG-FA. The synthetic route of Pasp-hyd-PEG-FA is show in Supplementary Figure 1. Briefly, folic acid (1 g, 2.27 mmol) was dissolved in 20 mL of dimethyl sulfoxide (DMSO), and NHS (0.5 g, 4.35 mmol) and EDCI (0.9 g, 4.71 mmol) were added. The reaction mixture was stirred under the protection of nitrogen for 12 h at room temperature. Then the reaction mixture was dropwise added into 10 mL of DMSO containing H2N-PEG-NH2 (4 g, 1 mmol). The reaction mixture was continuously stirred under the protection of nitrogen at room temperature for 2 h. Next, the reaction mixture was diluted with water and filtered. The filtrate was dialyzed against deionized water for 3 days by using a dialysis bag (molecular weight cutoff: 1 kDa). The solution in the dialysis bag was lyophilized by using a freeze dryer. Finally, FA-PEG-NH2 was further purified through an LH20 Sephadex column (Pharmacia, Uppsala, Sweden). 4-Acetylbenzoic acid (ABA, 0.1 g, 0.56 mmol), EDCI (0.15 g, 0.78 mmol), and NHS (0.09 g, 0.78 mmol) were dissolved in 5 mL of DMSO solution. The mixture was stirred at room 747

DOI: 10.1021/acs.molpharmaceut.6b01016 Mol. Pharmaceutics 2017, 14, 746−756

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Molecular Pharmaceutics Table 1. Characteristics of Pasp-hyd-PEG-FA Micellesa drug loading (%) molar ratio of Pasp:PEG 1:1 (blank) 1:1 (DOX) 1:1 (TPP−DOX) 1:1 (DOX/TPP−DOX) 1:4 (blank) 1:4 (DOX) 1:4 (TPP−DOX) 1:4 (DOX/TPP−DOX) 1:10 (blank) 1:10 (DOX) 1:10 (TPP−DOX) 1:10 (DOX/TPP−DOX) a

particle size (nm) 330 340 337 350 233 235 235 244 188 190 190 193

± ± ± ± ± ± ± ± ± ± ± ±

10 14 16 16 14 15 8 10 9 14 6 8

PDI 0.24 0.25 0.27 0.23 0.20 0.17 0.19 0.21 0.12 0.16 0.15 0.13

± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.01 0.02 0.02 0.02 0.01 0.01 0.03 0.01 0.03 0.02 0.01

zeta potential (mV) −8.9 −9.7 −7.6 −8.8 −15.9 −17.3 −14.7 −15.0 −18.6 −23.8 −15.7 −20.5

± ± ± ± ± ± ± ± ± ± ± ±

0.5 0.6 0.3 0.2 0.6 0.3 0.3 0.2 0.1 0.2 0.3 0.2

DOX

TPP−DOX

encapsulation efficiency (%) DOX

TPP−DOX

CMC (μg/mL) 0.5 ± 0.3

15.1 ± 0.7 10.6 ± 0.5

89 ± 4 11.7 ± 0.3 7.7 ± 0.4

85 ± 2

91 ± 2 88 ± 3 0.5 ± 0.2

13.8 ± 0.6 9.4 ± 0.7

87 ± 5 12.5 ± 0.2 8.2 ± 0.5

82 ± 3

88 ± 2 87 ± 3 0.4 ± 0.1

12.5 ± 0.3 9.9 ± 0.4

83 ± 2 14.2 ± 0.2 12.4 ± 0.3

84 ± 5

90 ± 3 85 ± 3

Data are presented as mean ± SD, n = 3.

was performed by model of multiple reaction monitoring (MRM). The mass spectrum conditions for MRM analysis are shown in Supplementary Table 1. A typical high performance liquid chromatogram of free DOX and TPP−DOX conjugate is shown in Supplementary Figure 2. 2.6. In Vitro Cytotoxicity of DOX/TPP−DOX@Pasphyd-PEG-FA. The cytotoxicity of free DOX, free TPP−DOX, blank micelles, DOX@Pasp-hyd-PEG-FA, TPP−DOX@Pasphyd-PEG-FA, and DOX/TPP−DOX@Pasp-hyd-PEG-FA against KB cells, MCF-7 cells, and MCF-7/ADR cells was evaluated by the MTT method. Briefly, the cells were planted into 96-well plates and cultured for 24 h. The culture medium was removed, and fresh culture medium that contained different drug formulation was added and incubated for 48 h. Thereafter, the culture medium was removed, and the wells were washed three times with PBS. The cells were cultured with fresh medium containing MTT (5 mg/mL) for 4 h. The culture medium was removed, and 150 μL of dimethyl sulfoxide (DMSO) was added into the well to dissolve the crystal. The absorbance was measured at 490 nm by using a Bio-Rad Microplate Reader (Richmond, CA, USA).13 The effect of folate on the cytotoxicity of DOX@Pasp-hyd-PEG-FA on MCF-7 cells was investigated by the same method. 2.7. Determination of Cleaved Caspase-3 Activity and Mitochondrial Membrane Potential. The effect of free DOX, free TPP−DOX, DOX@Pasp-hyd-PEG-FA, TPP− DOX@Pasp-hyd-PEG-FA, and DOX/TPP−DOX@Pasp-hydPEG-FA on the activity of cleaved caspase-3 level in tumor cells was detected by using caspase-3 detection kit (Beyotime Institute of Biotechnology, Jiangsu province, China).23 Briefly, KB cells and MCF-7/ADR cells were seeded in dishes and cultured for 24 h. The culture medium was removed, and fresh culture medium containing 10 μmol of DOX/L of free DOX, TPP−DOX, DOX@Pasp-hyd-PEG-FA, TPP−DOX@Pasphyd-PEG-FA, and DOX/TPP−DOX@Pasp-hyd-PEG-FA were added, respectively. After 24 h, cells were collected and resuspended in pyrolysis liquid. After that, the supernatant was collected by centrifuging for 15 min at 4 °C. Finally, 40 μL of supernatant and 10 μL of Ac-DEVD-pNA (2 mmol/L) were added to a 96-well plate, respectively. After 12 h incubation, the absorbance at 405 nm was determined by using a Bio-Rad Microplate Reader. The mitochondria-selective dye JC-1 was used to detect mitochondrial membrane potential. When JC-1 bonds with

high membrane potential mitochondria, it exhibits red fluorescence (590 nm). When JC-1 bonds with low membrane potential mitochondria, it exhibits green fluorescence (530 nm). KB cells and MCF-7/ADR cells were plated in a 6-well plate (1 × 106 cells per well) for 12 h. The culture medium was removed, the cells were cultured with free DOX and DOX/ TPP−DOX@Pasp-hyd-PEG-FA (the equivalent DOX concentration was 2 μmol/L) for 4 h, and then the cells were cultured with JC-1 solution (5 μg/mL) for 15 min at 37 °C and rinsed twice with assay buffer. The green fluorescent intensity of cell solution at 485/530 nm (excitation/emission wavelength) and red fluorescence at 530/590 nm were detected by using a fluorescence spectrophotometer. The ratio of red to green fluorescent intensity was calculated for each sample. 2.8. Subcellular Localization of DOX in Tumor Cells. KB cells and MCF-7/ADR cells were plated on glass coverslips and cultured for 24 h. The culture medium was removed, and the cells were cultured with the fresh medium containing DOX or DOX/TPP−DOX@Pasp-hyd-PEG-FA (2 μmol of DOX/L). After incubation for 4 h, the culture medium was removed. DAPI (10 μg/mL) containing medium was added and incubated with cells for 10 min. The cells were rinsed with PBS three times. The cells were then cultured with MitoTracker green (50 nmol/L) at 37 °C for 30 min. The culture medium was then removed, and cells were slightly washed with PBS three times. Finally, cells were fixed for 15 min by using formaldehyde. Confocal laser scanning microscopy (CLSM, Zeiss 510 LSMNLO confocal microscope, Jena, Germany) was used to observe the subcellular distribution of DOX. The effect of folate on cellular uptake of drug loaded micelles in MCF-7 cells was investigated by the same method by using the Olympus FV10-ASW (Tokyo, Japan). 2.9. Animal Experiment. MCF-7/ADR cells were subcutaneously implanted in the front right flank of female athymic nude mice (1 × 107 cells/0.1 mL/animal, 5 mice in each group). The mice were 6 weeks old, and their body weight was 20−22 g. Treatment was performed when the tumor size grew up to about 90 mm3. Free DOX (10 μmol/kg) or DOX/ TPP−DOX@Pasp-hyd-PEG-FA (2.0, 10 μmol/kg equivalent DOX) was injected to tumor-bearing mice by tail vein every fifth day (days 1, 5, and 10). Body weight and tumor size of tumor-bearing mice were recorded every 3 days.24 To study the biodistribution of DOX in tumor-bearing mice, free DOX (2 μmol/kg) and DOX/TPP−DOX@Pasp-hyd748

DOI: 10.1021/acs.molpharmaceut.6b01016 Mol. Pharmaceutics 2017, 14, 746−756

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Figure 1. TEM image, particle size distribution, and the stability of DOX/TPP−DOX@Pasp-hyd-PEG-FA. The molar ratio of Pasp to PEG was 1:1 in panel A, panel B, and panel C. The molar ratio of Pasp to PEG was 1:4 in panel D, panel E, and panel F. The molar ratio of Pasp to PEG was 1:10 in panel G, panel H, and panel I. Data are mean ± SD, n = 3.

PEG-FA (2 μmol of DOX/kg and 10 μmol of DOX/kg) were injected to the tumor-bearing mice by the tail vein. Tumorbearing mice were sacrificed at 12 or 24 h after the injection of drug, and the tumor tissues, lung, liver, spleen, kidney, and heart were collected. The fluorescence intensity in tumor tissues and different organs was detected by using an in vivo image system (Caliper IVIS Lumina II, Caliper Life Science, USA).24 Living Image 4.2 software was used to quantitatively analyze the fluorescence intensity in tumor tissues and organs. 2.10. Statistical Analysis. One-way ANOVA analysis in Sigma-Plot 8.0 software was used to compare different groups, and p < 0.05 was considered significantly difference.

micelles appeared spherical, and particle size distribution was generally uniform. All micelles were negatively charged. The particle size reduced with the increase of molar content of PEG. The TPP−DOX loading increased and the shape of the peak of particle size distribution became sharp with the increase of molar content of PEG. The molar ratio between Pasp and PEG showed no significant effect on DOX loading, encapsulation efficiency, and CMC. When molar ratio between Pasp and PEG was 1:10, the particle size of DOX/TPP−DOX@Pasp-hyd-PEG-FA was 193 nm, the drug loading for DOX and TPP−DOX was 9.9% and 12.4% respectively, and the encapsulation efficiency for DOX and TPP−DOX was 84% and 85% respectively. The above results indicated that, when the molar ratio between Pasp and PEG was 1:10, Pasp-hyd-PEG-FA was an ideal amphiphilic material to coencapsulate DOX and TPP−DOX. The size and PDI of DOX/TPP−DOX@Pasp-hyd-PEG-FA did not change in pH 7.4 medium containing 10% bovine serum albumin within 30 days, which implied that micelles could be stable in blood circulation. This may be due to the negative charge of the micelles and PEG moiety on the shell of micelles because negatively charged micelles could reduce its adsorption with plasma protein as well as its nonspecific cell adhering.27 The above data indicated that DOX/TPP−DOX@ Pasp-hyd-PEG-FA was a satisfactory drug delivery system. 3.3. Drug Release in Vitro. The drug release of DOX/ TPP−DOX@Pasp-hyd-PEG-FA in pH 7.4, pH 6.5, and pH 5.0 medium is shown in Figure 2. The amount of released DOX and TPP−DOX from DOX/TPP−DOX@Pasp-hyd-PEG-FA increased with the increase of molar content of PEG. The cumulative release rate of DOX and TPP−DOX from DOX/ TPP−DOX@Pasp-hyd-PEG-FA exhibited a pH-dependent manner. DOX/TPP−DOX@Pasp-hyd-PEG-FA released much more DOX and TPP−DOX in pH 5.0 medium than in pH 7.4 medium. When the molar ratio between Pasp and PEG was 1:10, more than 50% of loaded DOX and TPP−DOX were

3. RESULTS AND DISCUSSION 3.1. Characterization of Pasp-hyd-PEG-FA. The 1H NMR spectrum of Pasp-hyd-PEG-FA is presented in Supplementary Figure 3. The peaks at 7.1 and 7.8 ppm in 1H NMR spectrum indicated FA was connected with PEG. The signal at 3.6 ppm indicated that the PEG backbone existed the in Pasp-hyd-PEG-FA molecule. The signal at 4.6−4.7 ppm indicated the Pasp backbone. 3.2. Characterization of Drug Loaded Micelle. Biocompatibility, stability, particle size, and drug loading are key factors that need to be taken into consideration when we design and prepare the polymeric micelles. Particle size plays an important role in deciding the biodistribution of the micelle in vivo.25 Drug loading is closely related to the drug delivery efficiency in micelles. The stability of micelles affects the behavior in the bloodstream.26 The drug loading, encapsulation efficiency, particle size, polydispersity index, zeta potential, and critical micelle concentration (CMC) of the DOX/TPP−DOX@Pasp-hydPEG-FA are shown in Table 1. The TEM image, particle size distribution, and stability of DOX/TPP−DOX@Pasp-hydPEG-FA in PBS medium (pH 7.4, containing 10% bovine serum albumin) are shown in Figure 1. The shape of the 749

DOI: 10.1021/acs.molpharmaceut.6b01016 Mol. Pharmaceutics 2017, 14, 746−756

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Molecular Pharmaceutics

Figure 2. Release profiles of DOX and TPP−DOX from DOX/TPP−DOX@Pasp-hyd-PEG-FA in different pH release medium. The molar ratio of Pasp to PEG was 1:10 in panel A and panel B. The molar ratio of Pasp to PEG was 1:4 in panel C and panel D. The molar ratio of Pasp to PEG was 1:1 in panel E and panel F. Data are mean ± SD, n = 3.

micelles had none of the cytotoxicity on KB cells and MCF-7/ ADR cells. The cytotoxicity of DOX/TPP−DOX@Pasp-hyd-PEG-FA on KB cells, MCF-7 cells, and MCF-7/ADR cells is shown in Figure 3. The IC50 of DOX, TPP−DOX, DOX@Pasp-hydPEG-FA, TPP−DOX@Pasp-hyd-PEG-FA, and DOX/TPP− DOX@Pasp-hyd-PEG-FA on KB cells, MCF-7 cells, and MCF7/ADR cells is shown in Table 2. DOX/TPP−DOX@Pasphyd-PEG-FA exhibited dose-dependent cytotoxicity on KB cells, MCF-7 cells, and MCF-7/ADR cells. Compared with free DOX or TPP−DOX, the same dose of DOX@Pasp-hyd-PEGFA or DOX/TPP−DOX@Pasp-hyd-PEG-FA showed higher cytotoxicity on KB cells, MCF-7 cells, and MCF-7/ADR cells. Compared with DOX@Pasp-hyd-PEG-FA micelles, TPP− DOX@Pasp-hyd-PEG-FA micelles showed lower cytotoxicity on KB cells and MCF-7 cells. However, TPP−DOX@Pasphyd-PEG-FA showed greater cytotoxicity on MCF-7/ADR cells. DOX/TPP−DOX@Pasp-hyd-PEG-FA showed markedly higher cytotoxicity on MCF-7/ADR cells, MCF-7 cells, and KB cells as compared to DOX@Pasp-hyd-PEG-FA and TPP−

released from DOX/TPP−DOX@Pasp-hyd-PEG-FA in pH 5.0 medium in 8 h. This was because, at pH 5.0, the structure of the micelle was unstable due to the cleavage of the hydrazone bond between Pasp and PEG, which led to the disassembly of micelles and subsequently fast release of DOX and TPP−DOX from DOX/TPP−DOX@Pasp-hyd-PEG-FA. In pH 7.4 medium, the structure of the micelle was stable; only a small amount of drugs was released from micelles. The in vitro drug release characteristics implied that DOX/TPP−DOX@Pasphyd-PEG-FA could effectively delay the release of the entrapped drug in blood circulation, and the drug release could be accelerated in acidic organelles such as endolysosomes in tumor cells. Consequently, DOX/TPP−DOX@Pasp-hydPEG-FA could enhance drug delivery efficiency as well as increase therapeutic effect.28−31 3.4. In Vitro Cytotoxicity of DOX/TPP−DOX@Pasphyd-PEG-FA. The cytotoxicity of blank Pasp-hyd-PEG-FA micelles on KB cells and MCF-7/ADR cells is shown in Supplementary Figure 4. The results indicated that blank 750

DOI: 10.1021/acs.molpharmaceut.6b01016 Mol. Pharmaceutics 2017, 14, 746−756

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Molecular Pharmaceutics

Figure 3. Cytotoxicity of DOX/TPP−DOX@Pasp-hyd-PEG-FA on KB cells (left column), MCF-7 cells (medium column), and MCF-7/ADR cells (right column) in 48 h. The molar ratio of Pasp to PEG was 1:1 in panel A, panel B, and panel C. The molar ratio of Pasp to PEG was 1:4 in panel D, panel E, and panel F. The molar ratio of Pasp to PEG was 1:10 in panel G, panel H, and panel I. Data are mean ± SD, n = 3. **p < 0.01, *p < 0.05, vs TPP−DOX@Pasp-hyd-PEG-FA at the same concentration. ##p < 0.01, #p < 0.05, vs DOX@Pasp-hyd-PEG-FA at the same concentration.

MCF-7 cells was mediated by the folate receptor, and folate in micelles played an important role in the enhancement of uptake and cytotoxicity of FA modified micelles on folate receptor positive tumor cells. 3.5. The Cleaved Caspase-3 Activity. It was reported that DOX can induce mitochondrial dysfunction and cell apoptosis.32,33 The cleaved caspase-3 is the main apoptotic executive molecule. The cleaved caspase-3 level quantitatively reflects the extent of cell apoptosis. The cleaved caspase-3 level was detected by the cleaved caspase-3 activity assay kit. When KB cells and MCF-7/ADR cells were cultured with free DOX, free TPP−DOX, and DOX/TPP−DOX@Pasp-hyd-PEG-FA for 24 h, the cleaved caspase level in KB cells and MCF-7/ADR cells was as shown in Figures 5A and 5B, respectively. Compared with free DOX, free TPP−DOX showed less effect on cleaved caspase-3 level in KB cells. On the other hand, free TPP−DOX significantly increased cleaved caspase-3 level in MCF-7/ADR cells as compared to free DOX. Compared with free DOX, DOX@Pasp-hyd-PEG-FA obviously increased cleaved caspase-3 level in KB cells and MCF-7/ADR cells. TPP−DOX@Pasp-hyd-PEG-FA significantly increased the level of cleaved caspase-3 in MCF-7/ ADR cells as compared with DOX@Pasp-hyd-PEG-FA. DOX/ TPP−DOX@Pasp-hyd-PEG-FA markedly increased the level of cleaved caspase-3 in KB cells and MCF-7/ADR cells as compared to TPP−DOX@Pasp-hyd-PEG-FA micelles. The above results were well consistent with the results of cytotoxicity of drug loaded micelles. These results also implied

Table 2. IC50 of DOX, TPP−DOX, DOX@Pasp-hyd-PEGFA, TPP−DOX@Pasp-hyd-PEG-FA, and DOX/TPP− DOX@Pasp-hyd-PEG-FA on KB Cells, MCF-7 Cells, and MCF-7/ADR Cellsa IC50 (μmol/L) KB DOX TPP−DOX DOX@Pasp-hyd-PEG-FA TPP−DOX@Pasp-hyd-PEG-FA DOX/TPP−DOX@Pasp-hydPEG-FA a

3.4 48.7 2.6 40.1 0.9

± ± ± ± ±

MCF-7 0.4 2.6 0.5 1.7 0.2

4.4 62.3 0.8 19.2 0.4

± ± ± ± ±

0.9 2.7 0.2 0.6 0.1

MCF-7/ ADR 87.6 32.8 15.9 7.2 0.2

± ± ± ± ±

3.7 1.5 1.6 0.8 0.1

Molar ratio of Pasp:PEG = 1:10, n = 3.

DOX@Pasp-hyd-PEG-FA. Furthermore, the cytotoxicity of DOX/TPP−DOX@Pasp-hyd-PEG-FA on KB cells, MCF-7 cells, and MCF-7/ADR cells enhanced with the increase of the molar content of PEG. This result was consistent with the reults of in vitro drug release. Thus, the micellar material in which the molar ratio of Pasp to PEG was 1:10 was used in the following experiment. The effect of folate on the cytotoxicity and cell uptake of DOX@Pasp-hyd-PEG-FA in MCF-7 cell was investigated, and the results are shown in Figure 4. The results indicated that the cytotoxicity and uptake of DOX@Pasp-hyd-PEG-FA in MCF-7 cells was significantly reduced in the presence of folate. Those results implied that the uptake of FA modified micelles by 751

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Figure 4. Effect of folate on the cytotoxicity (panel A) and cellular uptake (panel B) of DOX@Pasp-hyd-PEG-FA in MCF-7 cells. The concentration of folate was 10 μmol/L. The equivalent DOX concentration was 2 μmol/L in the cellular uptake experiment. The magnification of fluorescent microscopy was 60× oil immersion objective and 10× ocular lens. Data are mean ± SD, n = 3. *p < 0.05, vs DOX@Pasp-hyd-PEG-FA at the same concentration.

Figure 5. Cleaved caspase-3 activity in KB cells (A) and MCF-7/ADR cells (B) that were treated with DOX/TPP−DOX@Pasp-hyd-PEG-FA for 24 h. **p < 0.01, *p < 0.05, vs TPP−DOX@Pasp-hyd-PEG-FA. ##p < 0.01, #p < 0.05, vs DOX@Pasp-hyd-PEG-FA. The effect of DOX and DOX/ TPP−DOX@Pasp-hyd-PEG-FA on mitochondrial membrane potential of KB cells (C) and MCF-7/ADR cells (D). *p < 0.05, vs DOX. The equivalent DOX concentration was 2 μmol/L. Data are mean ± SD, n = 3.

not significantly decrease after MCF-7/ADR cells were cultured with DOX. But the ratio significantly decreased after MCF-7/ ADR cells were treated with DOX/TPP−DOX@Pasp-hydPEG-FA. Similar results were obtained on KB cells. The results implied that mitochondrial membrane potential in MCF-7/ ADR cell was significantly damaged by DOX/TPP−DOX@ Pasp-hyd-PEG-FA. The above data also indicated that DOX/ TPP−DOX@Pasp-hyd-PEG-FA induced the apotosis on KB cells and MCF-7/ADR cells through decreasing the mitochondrial membrane potential. 3.7. The Subcellular Localization of DOX. In order to further illustrate the mechanism of damage of mitochondrial membrane potential cuased by DOX/TPP−DOX@Pasp-hydPEG-FA, the subcellular localization of DOX in tumor cells was

that the enhanced cytotoxicity of DOX/TPP−DOX@Pasphyd-PEG-FA on MCF-7/ADR cells was due to the increase of the apoptosis level in DOX/TPP−DOX@Pasp-hyd-PEG-FA treated MCF-7/ADR cells. 3.6. The Effect of DOX/TPP−DOX@Pasp-hyd-PEG-FA on Mitochondrial Membrane Potential. When DOX accumulates in the mitochondria, it can damage the mitochondrial membrane potential, which results in the shedding of cytochrome c from mitochondria, then activation of caspase 3, and eventually induces tumor cell apoptosis.34−37 Thus, the effect of DOX/TPP−DOX@Pasp-hyd-PEG-FA on the mitochondrial membrane potential was investigated. As shown in Figures 5C and 5D, the ratio between red fluorescence intensity and green fluorescence intensity did 752

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about −180 to −200 mV.41 This high negative membrane potential is not present in any other cellular organelle, which offers a unique chemical opportunity for lipophilic cations to selectively accumulate in the mitochondria.42 Several lipophilic cations including TPP and rhodamine 123 were proved to selectively accumulate in the mitochondria.43 Owing to its delocalized cationic charge and lipophilicity, TPP modified conjugate can penetrate the lipophilic barrier of mitochondrial membrane.44 Some bioactive molecules including antioxidants have been conjugated with TPP to selectively deliver them to mitochondria.45 For example, the TPP has been conjugated with vitamin E. The results demonstrated that TPP−vitamin E conjugate increased the accumulation of vitamin E in the mitochondria and more effectively attenuated the oxidative damage of mitochondria than vitamin E did.46 In addition, mitochondria-target MitoQ, a TPP-conjugated ubiquinone derivative, has been used extensively to prevent diseases associated with mitochondrial oxidative stress.47,48 MitoQ accumulated more than 100-fold in mitochondria and shows greater efficacy than the nontargeted CoQ10 analogue did.49 Besides connection with small molecule drug, TPP has also been used to modify the drug carriers to deliver anticancer agents to mitochondria. This kind of mitochondria-specific drug delivery system includes TPP-polyethylene imine (TPPPEI) modified nanoparticle (loaded with DOX),50 TPP-stearyl modified liposome (loaded with DOX),51 poly(lactide-coglycolide)-b-poly(ethylene glycol)-TPP (PLGA-b-PEG-TPP) modified nanoparticles (loaded with lonidamine),52 and TPPpoly(ethylene glycol)-phosphatidylethanolamine (TPP-PEGPE) modified liposomes (loaded with paclitaxel).53,54 In theory, compared with the above TPP modified nanocarriers, it is much easier for TPP−DOX to penetrate the mitochondria membrane because TPP−DOX conjugate is a small molecule. However, TPP−DOX conjugate lacks selectivity for tumor tissue. In this study, DOX and TPP−DOX were coentrapped by Pasp-hydPEG-FA micelles. DOX/TPP−DOX@Pasp-hyd-PEG-FA can be accumulated in tumor tissue by the enhanced permeability and retention effect and, subsequently, be taken up by tumor cells through FA receptor mediated endocytosis. Finally, DOX and TPP−DOX diffuse to nucleus and mitochondria to induce cell apoptosis. 3.8. Antitumor Activity of DOX/TPP−DOX@Pasp-hydPEG-FA in Vivo. The antitumor activity of DOX/TPP− DOX@Pasp-hyd-PEG-FA in vivo is shown in Figures 7A and 7B. The tumor volume is a gold index to evaluate the antitumor activity. The tumor volume quickly increased in tumor-bearing mice that were treated with normal saline. The tumor volume in the normal saline treated group was about 1200 mm3 at the end of the experiment. The tumor volume in the 10 μmol/kg free DOX treated group was about 650 mm3 at the end of the experiment. However, the tumor volume was significantly shrunk in a dose-dependent manner in tumor-bearing mice that were treated with DOX/TPP−DOX@Pasp-hyd-PEG-FA. The tumor volume in the 10 μmol DOX/kg DOX/TPP−DOX@ Pasp-hyd-PEG-FA treated group was about 200 mm3 at the end of the experiment. Body weight is an objective index of systemic toxicity. The body weight gradually decreased in the group of tumor-bearing mice that were treated with free DOX, and the body weight gradually increased in the group of tumorbearing mice that were treated with DOX/TPP−DOX@Pasphyd-PEG-FA. Thus, the above results indicate that DOX/ TPP−DOX@Pasp-hyd-PEG-FA enhanced the antitumor activ-

investigated by CLSM. As shown in Figures 6A and 6B, when DOX/TPP−DOX@Pasp-hyd-PEG-FA was incubated with KB

Figure 6. CLSM image of KB cells treated with DOX@Pasp-hydPEG-FA (A) and DOX/TPP−DOX@Pasp-hyd-PEG-FA (B) at 37 °C for 4 h. 60× oil immersion objective and 10× ocular lens. CLSM image of MCF-7/ADR cells treated with DOX@Pasp-hyd-PEG-FA (C) and DOX/TPP−DOX@Pasp-hyd-PEG-FA (D), 60× oil immersion objective and 10× ocular lens. The DOX concentration was 2 μmol/L. The yellow color indicates the localization of DOX (red) in mitochondria (green). The pink region indicates the localization of DOX (red) in the nucleus (blue).

cells for 4 h, a great amount of DOX distributed in mitochondria and nucleus. This was the reason that DOX/ TPP−DOX@Pasp-hyd-PEG-FA induced more apoptosis and showed higher cytotoxicity on KB cells than DOX@Pasp-hydPEG-FA did. As shown in Figures 6C and 6D, when DOX@ Pasp-hyd-PEG-FA was incubated with MCF-7/ADR cells for 4 h, a small amount of DOX distributed in the cytoplasm and nucleus. This was the reason that DOX@Pasp-hyd-PEG-FA showed low cytotoxicity on MCF-7/ADR cells. When DOX/ TPP−DOX@Pasp-hyd-PEG-FA was incubated with MCF-7/ ADR cells for 4 h, a large amount of DOX distributed in the mitochondria, and some amount of DOX distributed in the nucleus, consequently leading to great damage of mitochondrial membrane potential and significant cytotoxicity on MCF-7/ ADR cells. The above results indicated that there existed significant difference in the uptake of DOX between wild type tumor cells and DOX-resistant tumor cells. Compared with wild type tumor cells, the uptake of DOX in DOX-resistant tumor cells was markedly decreased. MCF-7/ADR cells were generated by culturing MCF-7 cells with low concentration DOX in a long time. One exact mechanism by which MCF-7 cells show resistance to DOX is the overexpression of p-glycoprotein (Pgp), which decreases the accumulation of DOX in MCF-7 cells.38,39 TPP−DOX could accumulate in KB cells and MCF7/ADR cells, which implies that TPP−DOX was not a substrate of P-gp. Generally, DOX shows high affinity to DNA in the nucleus. However, multiple interactions exist between mitochondria and DOX, which induce mitochondrial dysfunction and the apoptosis of cells.40 The mitochondrial membrane potential is 753

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Figure 7. Antitumor activity of DOX/TPP−DOX@Pasp-hyd-PEG-FA in vivo. Panel A shows the body weight changes in tumor-bearing nude mice. Panel B shows the tumor volume changes in tumor-bearing mice. Panel C shows the DOX distribution in the organs and tumor tissue of tumorbearing nude mice at 12 and 24 h after intravenous injection of free DOX or DOX/TPP−DOX@Pasp-hyd-PEG-FA. Panel D shows the semiquantitative analysis of fluorescence intensity of DOX in tumor tissue and organs. Data are mean ± SD, n = 5.

3.9. Drug Biodistribution. After the injection of free DOX or DOX/TPP−DOX@Pasp-hyd-PEG-FA by tail vein, the

ity in a DOX-resistant tumor without causing obvious systemic toxicity. 754

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(7) Han, M.; Vakili, M. R.; Soleymani Abyaneh, H.; Molavi, O.; Lai, R.; Lavasanifar, A. Mitochondrial Delivery of Doxorubicin via Triphenylphosphine Modification for Overcoming Drug Resistance in MDA-MB-435/DOX Cells. Mol. Pharmaceutics 2014, 11, 2640. (8) Aschenbrenner, A. J.; Balota, D. A.; Fagan, A. M.; Duchek, J. M.; Benzinger, T. L.; Morris, J. C. Alzheimer Disease Cerebrospinal Fluid Biomarkers Moderate Baseline Differences and Predict Longitudinal Change in Attentional Control and Episodic Memory Composites in the Adult Children Study. J. Int. Neuropsychol. Soc. 2015, 21, 573. (9) Taveira, S. F.; de Campos Araújo, L. M. P.; de Santana, D. C.; Nomizo, A.; de Freitas, L. A.; Lopez, R. F. Development of cationic solid lipid nanoparticles with factorial design-based studies for topical administration of doxorubicin. J. Biomed. Nanotechnol. 2012, 8, 219. (10) Kim, J. H.; Li, Y.; Kim, M. S.; Kang, S. W.; Jeong, J. H.; Lee, D. S. Synthesis and evaluation of biotin-conjugated pH-responsive polymeric micelles as drug carriers. Int. J. Pharm. 2012, 427, 435. (11) Pujade-Lauraine, E.; Wagner, U.; Aavall-Lundqvist, E.; Gebski, V.; Heywood, M.; Vasey, P. A.; Volgger, B.; Vergote, I.; Pignata, S.; Ferrero, A.; Sehouli, J.; Lortholary, A.; Kristensen, G.; Jackisch, C.; Joly, F.; Brown, C.; Le Fur, N.; du Bois, A. Pegylated liposomal Doxorubicin and Carboplatin compared with Paclitaxel and Carboplatin for patients with platinum-sensitive ovarian cancer in late relapse. J. Clin. Oncol. 2010, 28, 3323. (12) Tan, L.; Neoh, K. G.; Kang, E. T.; Choe, W. S.; Su, X. PEGylated anti-MUC1 aptamer-doxorubicin complex for targeted drug delivery to MCF7 breast cancer cells. Macromol. Biosci. 2011, 11, 1331. (13) Ye, W. L.; Du, J. B.; Zhang, B. L.; Na, R.; Song, Y. F.; Mei, Q. B.; Zhao, M. G.; Zhou, S. Y. Cellular uptake and antitumor activity of DOX-hyd-PEG-FA nanoparticles. PLoS One 2014, 9, e97358. (14) Ye, W. L.; Teng, Z. H.; Liu, D. Z.; Cui, H.; Liu, M.; Cheng, Y.; Yang, T. H.; Mei, Q. B.; Zhou, S. Y. Synthesis of a new pH-sensitive folate-doxorubicin conjugate and its antitumor activity in vitro. J. Pharm. Sci. 2013, 102, 530. (15) Yoo, H. S.; Park, T. G. Folate-receptor-targeted delivery of doxorubicin nano-aggregates stabilized by doxorubicin-PEG-folate conjugate. J. Controlled Release 2004, 100, 247. (16) Hwa Kim, S.; Hoon Jeong, J.; Joe, C. O.; Gwan Park, T. Folate receptor mediated intracellular protein delivery using PLL-PEG-FOL conjugate. J. Controlled Release 2005, 103, 625. (17) Ghaghada, K. B.; Saul, J.; Natarajan, J. V.; Bellamkonda, R. V.; Annapragada, A. V. Folate targeting of drug carriers: a mathematical model. J. Controlled Release 2005, 104, 113. (18) Thapa, R. K.; Choi, Y.; Jeong, J. H.; Youn, Y. S.; Choi, H. G.; Yong, C. S.; Kim, J. O. Folate-Mediated Targeted Delivery of Combination Chemotherapeutics Loaded Reduced Graphene Oxide for Synergistic Chemo-Photothermal Therapy of Cancers. Pharm. Res. 2016, 33, 2815. (19) Fasehee, H.; Dinarvand, R.; Ghavamzadeh, A.; EsfandyariManesh, M.; Moradian, H.; Faghihi, S.; Ghaffari, S. H. Delivery of disulfiram into breast cancer cells using folate-receptor-targeted PLGA-PEG nanoparticles: in vitro and in vivo investigations. J. Nanobiotechnol. 2016, 14, 32. (20) Du, J. B.; Song, Y. F.; Ye, W. L.; Cheng, Y.; Cui, H.; Liu, D. Z.; Liu, M.; Zhang, B. L.; Zhou, S. Y. PEG-detachable lipid-polymer hybrid nanoparticle for delivery of chemotherapy drugs to cancer cells. Anti-Cancer Drugs 2014, 25, 751. (21) Sawant, R. R.; Torchilin, V. P. Enhanced cytotoxicity of TATpbearing paclitaxel-loaded micelles in vitro and in vivo. Int. J. Pharm. 2009, 374, 114. (22) Qiu, L. Y.; Bae, Y. H. Self-assembled polyethylenimine-graftpoly(epsilon-caprolactone) micelles as potential dual carriers of genes and anticancer drugs. Biomaterials 2007, 28, 4132. (23) Hann, S. S.; Tang, Q.; Zheng, F.; Zhao, S.; Chen, J.; Wang, Z. Repression of phosphoinositide-dependent protein kinase 1 expression by ciglitazone via Egr-1 represents a new approach for inhibition of lung cancer cell growth. Mol. Cancer 2014, 13, 149. (24) Du, J. B.; Cheng, Y.; Teng, Z. H.; Huan, M. L.; Liu, M.; Cui, H.; Zhang, B. L.; Zhou, S. Y. pH-Triggered Surface Charge Reversed

biodistribution of DOX in tumor-bearing mice is shown in Figures 7C and 7D. DOX was typically accumulated in heart, liver, lung, spleen, kidney, and tumor tissue after the adminstration of free DOX. However, DOX/TPP−DOX@ Pasp-hyd-PEG-FA obviously increased the accumulation of DOX in the tumor tissue, and significantly reduced the accumulation of DOX in heart, liver, lung, spleen, and kidney, which resulted in the significant enhancement of antitumor activity of DOX/TPP−DOX@Pasp-hyd-PEG-FA in DOXresistant tumor and the decrease of systemic toxicity of DOX.

4. CONCLUSION DOX/TPP−DOX@Pasp-hyd-PEG-FA delivered DOX and TPP−DOX to nucleus and mitochondria of tumor cells simultaneously and consequently induced more apoptosis on wild-type tumor cells and DOX-resistant tumor cells. Thus, the antitumor activity of DOX/TPP−DOX@Pasp-hyd-PEG-FA on DOX-resistant tumor was significantly enhanced in vivo. Mitochondria and nucleus dual delivery system showed great potential in treatment of DOX-resistant tumor.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b01016. Synthetic scheme and HPLC, 1H NMR, and cytotoxicity results (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Changle West Road 169, Shaanxi province, Xi’an, 710032, China. E-mail: [email protected]. ORCID

Si-yuan Zhou: 0000-0001-6092-4406 Author Contributions ‡

H.C. and M.-l.H. equally contributed to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by Science and Technology Foundation of Shaanxi (2012KTCL03-18). REFERENCES

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