Micelle System Based on Molecular Economy Principle for

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Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Micelle System Based on Molecular Economy Principle for Overcoming Multidrug Resistance and Inhibiting Metastasis Yan Qi,†,‡ Xianya Qin,† Conglian Yang,† Tingting Wu,† Qi Qiao,† Qingle Song,† and Zhiping Zhang*,†,§,∥ †

Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan 430030, P.R. China Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, Sichuan University, Chengdu 610065, P.R. China § National Engineering Research Center for Nanomedicine, Huazhong University of Science and Technology, Wuhan 430074, P.R. China ∥ Hubei Engineering Research Centre for Novel Drug Delivery System, Huazhong University of Science and Technology, Wuhan 430030, P.R. China ‡

S Supporting Information *

ABSTRACT: The high mortality of cancer is mainly attributed to multidrug resistance (MDR) and metastasis. A simple micelle system was constructed here to codeliver doxorubicin (DOX), adjudin (ADD), and nitric oxide (NO) for overcoming MDR and inhibiting metastasis. It was devised based on the “molecular economy” principle as the micelle system was easy to fabricate and exhibited high drug loading efficiency, and importantly, each component of the micelles would exert one or more active functions. DOX acted as the main cell killing agent supplemented with ADD, NO, and D-αtocopheryl polyethylene glycol 1000 succinate (TPGS). MDR was overcome by synergistic effects of mitochondria inhibition agents, TPGS and ADD. A TPGS-based NO donor can be used as a drug carrier, and it can release NO to enhance drug accumulation and penetration in tumor, resulting in a positive cycle of drug delivery. This DOX−ADD conjugate self-assembly system demonstrated controlled drug release, increased cellular uptake and cytotoxicity, enhanced accumulation at tumor site, and improved in vivo metastasis inhibition of breast cancer. The micelles can fully take advantage of the functions of each component, and they provide a potential strategy for nanomedicine design and clinical cancer treatment. KEYWORDS: D-α-tocopheryl polyethylene glycol 1000 succinate, multidrug resistance, metastasis, drug delivery, breast cancer

1. INTRODUCTION Breast cancer is now the most common cancer in females.1 From Cancer Statistics 2017, there would be about 252 710 patients diagnosed with breast cancer in United States, accounting for 30% of the new cancer cases of females.2 Breast cancer affects the life of women worldwide. The high mortality of breast cancer is mainly attributed to the metastasis and multidrug resistance (MDR).3 At advanced stages, breast cancer preferentially metastasizes to distant organs, making it fatal and incurable.4 Metastasis, a complicated process, is related to the interactions between cancer cells and microenvironment of the invaded organs.5 It may be also correlated with MDR.6 MDR, a commonly reported phenomenon in cancer therapy, should be responsible for the failure of clinical cancer treatment. It could be caused by various mechanisms, including increased drug efflux, decreased drug influx, and varied drug metabolism.7 Drug efflux, one of the predominant causes in MDR, is related to membrane-mediated drug transporters, in which the most well investigated is P-glycoprotein (P-gp).8,9 Substrate drugs can be pumped out by P-gp, thus, reducing intracellular drug © XXXX American Chemical Society

accumulation and restricting cytotoxic effect of chemotherapeutics in cancer treatment. Therefore, the therapy aiming at metastasis and MDR inhibition of breast cancer is highly desired. As a commonly used chemotherapeutic agent in breast cancer, doxorubicin (DOX) is confronted with some challenges, including the rapid clearance during circulation, severe side effects, and complicated MDR.10 It has been demonstrated that nanotechnology can provide a potential platform for chemotherapeutic drugs delivery by improving blood circulation, enhancing drug accumulation in tumor, and reducing side effects.11 Moreover, codelivering DOX and P-gp related inhibitors by nanomedicine has been a promising strategy to inhibit metastasis and overcome MDR of breast cancer.12−14 Through disrupting the energy supply of Received: Revised: Accepted: Published: A

October 23, 2017 January 11, 2018 February 4, 2018 February 4, 2018 DOI: 10.1021/acs.molpharmaceut.7b00922 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Scheme 1. Schematic Illustration of the Preparation and Multimechanisms of Synergistic Antitumor Effects of DOX−ADD@ TPGS−NO Micelles

NO may provide a promising therapeutic regimen for reversing MDR of DOX in breast cancer. In this work, we constructed DOX−ADD@TPGS−NO micelles based on the “molecular economy” principle. The system is very simple and can fully take advantage of the functions of each component (Scheme 1). The “molecular economy” was supposed to describe the fact that, (1) the fabrication of micelles was quite facile, which saved time and energy; (2) the micelles exhibited high drug loading efficiency, which meant less consumption of drugs or excipients; and (3) more importantly each component in the micelle system was supposed to exhibit one or more active functions. The TPGSderived NO donor can act as the drug delivery carrier to promote self-assembly of pH-sensitive DOX−ADD prodrug. Besides, TPGS itself has shown great potential in MDR reversal and metastasis suppression. The redox-sensitive NO donor can efficiently deliver NO and enhance drug accumulation and penetration. DOX is the main agent for tumor cells killing. ADD can disrupt the mitochondria function to inhibit P-gp activity and kill tumor cells. The micelles were simply fabricated by a thin-film hydration method. In vitro cell uptake and cytotoxicity of DOX−ADD@TPGS−NO micelles were studied on drug-sensitive (MCF7) and drug-resistant human breast cancer cells (MCF7/ADR). The associated mechanisms of overcoming MDR by TPGS and ADD were investigated, including ROS generation, mitochondrial membrane potential change, and intracellular ATP level. In vivo pharmacokinetics and biodistribution were further conducted to assess the blood

mitochondria, the inhibitor can efficiently influence the function of ATP-dependent efflux transporter P-gp, and thus, overcome MDR. Adjudin (ADD), a male contraceptive with the function of mitochondria inhibition, was reported to serve as a mitochondria-targeted anticancer drug.15,16 The in vitro activity of ADD has already been investigated on drug resistant cells in our previous work.17 D-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) is extensively applied in drug delivery.18 There is a mounting of drug delivery systems based on TPGS, with the characteristics of high-loading efficiency, controlled drug release profiles, and enhanced therapeutic efficacy.10,19 Besides, TPGS can exert inhibition effect on P-gp through inducing mitochondria dysfunction, as indicated by the reactive oxygen species (ROS) generation, mitochondria membrane potential loss, and ATP level downregulation.20 TPGS also demonstrated great potential in inhibiting metastasis.21 Nitric oxide (NO), an endogenous gaseous molecule, has been reported to regulate angiogenesis and vasodilation.22 NO was supposed to boost the sensitivity of cancer cells to chemotherapeutics by normalizing tumor vasculature and improving oxygenation level at the tumor site, thus, enhancing the antitumor efficiency and overcoming MDR.23 To overcome the shortages of extreme short half-time and chemical instability of NO in clinical application, we previously synthesized a NO donor, nitrate-functionalized TPGS derivative (TNO3).24 Combination of NO donor and chemotherapeutics could enhance drug delivery at the tumor site and reverse MDR.25 It would be inferred that the combination of ADD, TPGS, and B

DOI: 10.1021/acs.molpharmaceut.7b00922 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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In vitro drug release of DOX−ADD@TPGS−NO micelles was also performed. NO release was conducted as previously reported.24 Briefly, DOX−ADD@TPGS−NO micelles in pH 7.4 PBS with or without 10 mM DTT were placed in a bath shaker at 37 °C, 100 rpm. At predetermined time points, 50 μL of the outer-release medium was collected, and fresh medium was supplemented. The released NO was determined using the traditional Griess assay.26 The DOX release experiment was evaluated by a dialysis method at 37 °C, which was carried out in two different buffer solutions of pH 5.0 acetate buffer solution (ABS) and pH 7.4 PBS. At the assigned intervals of 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, and 144 h, the outer phase was replaced with fresh buffer solution. DOX concentration was measured by HPLC, and the fluorescence detector was set at 470/585 nm (excitation/emission). All experiments were performed three times at each condition. 2.5. In Vitro Cellular Uptake of DOX−ADD@TPGS−NO Micelles. The cellular uptake of DOX−ADD@TPGS−NO micelles was investigated in MCF7 and MCF7/ADR cells. Cells/well (2.0 × 105) were seeded for overnight culture. Serum-free culture medium (control), free-DOX, DOX−ADD, or DOX−ADD@TPGS−NO micelles, with the equivalent DOX concentration of 5 μM, were incubated with cells for 1, 2, and 4 h, respectively. Then, the cells were treated by trypsinization, washed, and centrifuged to detect the cellular uptake by flow cytometer. For further visualization of cellular distribution, a confocal laser scanning microscope (CLSM, 710META, Zeiss, Germany) was applied. The cells were seeded into 24-well plates with 1.0 × 104 cells/well. After 24 h, the medium was substituted for blank medium, DOX, DOX−ADD, or DOX−ADD@TPGS−NO micelles with a DOX concentration of 5 μM. After 4 h, cells were stained with DAPI in the dark, and then fixed for visual observation of cellular distribution by CLSM. 2.6. Cytotoxicity of DOX−ADD@TPGS−NO Micelles. MCF7 and MCF7/ADR cells were seeded into 96-well plates with a density of 5.0 × 103 cells per well. With overnight adherence growth, cells were incubated with free-DOX, ADD, DOX−ADD, TPGS−NO, or DOX−ADD@TPGS−NO micelles, with DOX or NO concentration ranging from 0.02 to 20 μM (the molar ratio of DOX to ADD was 1:1), for 24, 48, or 72 h. MTT assay was used to evaluate the cell viability. The SPSS software (version 19.0) was adopted to calculate the halfmaximal inhibitory concentrations (IC50) values. 2.7. Synergistic Mechanism Study of Overcoming MDR by TPGS and ADD. ROS Production. The ROS production was determined by an assay kit (Beyotime Institute of Biotechnology, China). MCF7/ADR cells, with 2.0 × 105 cells per well, were cultured on a 6-well plate. After being incubated with blank medium, DOX, ADD, DOX−ADD, TPGS, TPGS−NO, or DOX−ADD@TPGS−NO micelles, with DOX or NO concentration of 5 μM (the molar ratio of DOX to ADD was 1:1), for 6 h, the cells were harvested and incubated with dichlorodihydrofluorescein diacetate (DCFHDA) at the concentration of 10 μM at 37 °C for 20 min. Then the DCF fluorescence was analyzed by flow cytometry. Mitochondrial Depolarization. MCF7/ADR cells, seeded at the density of 2.0 × 105 cells, were incubated for 6 h with blank medium, DOX, ADD, DOX−ADD, TPGS, TPGS−NO, or DOX−ADD@TPGS−NO micelles, with DOX or NO concentration of 5 μM (the molar ratio of DOX to ADD was 1:1). Afterward, the collected cells were determined with a JC-1

circulation and tumor accumulation of micelles, respectively. The micelles exhibited significant tumor growth and metastasis inhibition effects. Altogether, the DOX−ADD@TPGS−NO micelles provided an attractive strategy for nanomedicine design and clinical application in cancer treatment.

2. MATERIALS AND METHODS 2.1. Materials. ADD, with purity of 99%, was obtained from Candia Pharmatech Co., Ltd. Doxorubicin hydrochloride (DOX·HCl) was purchased from Huafeng United Technology Co., Ltd. (Beijing, China). TPGS, 4-bromobutyl chloride (4BrC, 95%), and silver nitrate (AgNO3, 99.8%) were bought from Sigma-Aldrich (St. Louis, USA). DL-Dithiothreitol (DTT) was obtained from Aladdin, China. 1,1′-Dioctadecyl-3,3,3′,3′tetramethylindotricarbocyanine iodide (DIR) was bought from AAT Bioquest Inc.(USA). Fetal bovine serum (FBS), RPMI 1640 and DMEM medium, penicillin-streptomycin, and trypsin were bought from Hyclone (USA). 4′,6-Diamidino-2-phenylindole (DAPI) and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Biosharp, South Korea. 2.2. Cell Culture. Drug-resistant MCF7/ADR cells, kindly provided by Dr. Yaping Li (Shanghai Institute of Materia Medica, Chinese Academy of Sciences), were cultured in complete RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin G sodium, and 100 μg/mL streptomycin sulfate with 1 μg/mL DOX. MCF7 cells were obtained from the Chinese Academy of Sciences Cells Bank (Shanghai, China) and cultivated in complete DMEM medium. The murine mammary carcinoma cell, 4T1, was offered by Dr. Ding Ma (Tongji Hospital of Huazhong University of Science and Technology). 4T1 cells were cultivated in complete Gibco 1640 medium with the condition of 5% CO2 and 37 °C. 2.3. Animals. Female Balb/c mice within the age of 4−5 weeks were purchased from the Disease Control Center of Hubei, and they were fed in the SPF grade condition at Tongji Animal Center. Female Kunming mice (18−20 g) and SpragueDawley rats (180−200 g) were purchased from the Tongji Animal Center of Huazhong University of Science and Technology. All animal experiments were conducted under the regulations and guidelines of the Ethics Committee of Huazhong University of Science and Technology. 2.4. Preparation and Characterization of DOX−ADD@ TPGS−NO Micelles. The DOX−ADD conjugate and TPGSbased NO donor (TPGS-NO) were synthesized according to our previous works.17,24 DOX−ADD@TPGS−NO micelles were fabricated by a thin-film hydration method. Briefly, 2 mg DOX−ADD and 16 mg TPGS−NO were dissolved in 5 mL of methanol, followed by rotary evaporation at 40 °C under reduced pressure. Then DOX−ADD@TPGS−NO micelles were obtained by hydration for 30 min in a bath shaker at 37 °C with 100 rpm. For the characterization of DOX−ADD@TPGS−NO micelles, dynamic light scattering (DLS, Zeta Plus, Brookhaven, USA) was used to measure particle size and size distribution. Transmission electron microscope (TEM, JEM-1230, Japan) was applied for the morphology observation. The particle size changes of micelles were monitored for 7 days in pH 7.4 phosphate buffer solution (PBS) and fatal bovine serum (FBS) for stability evaluation. The drug encapsulation efficiency was analyzed by high-performance liquid chromatography (HPLC) (UltiMate 3000 Thermo scientific), with the fluorescence detector set at 470 nm/585 nm (excitation/emission). C

DOI: 10.1021/acs.molpharmaceut.7b00922 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 1. Characterization of DOX−ADD@TPGS−NO micelles. (A) TEM image and size distribution of DOX−ADD@TPGS−NO micelles. (B) The stability of micelles during the storage in PBS and FBS for 7 days.

Figure 2. In vitro drug uptake of DOX−ADD@TPGS−NO micelles in MCF7 and MCF7/ADR cells. The cells were incubated with free-DOX, DOX−ADD, and DOX−ADD@TPGS−NO micelles. Then DOX accumulation was measured by a flow cytometer. The fluorescence of DOX in (A) MCF7 cells and (B) MCF7/ADR cells. CLSM images of (C) MCF7 cells and (D) MCF7/ADR cells after being treated with different formulations for 4 h. The red represents the signal of DOX, and the nucleus was stained by DAPI, which presented blue in the images.

at 0.5, 1, 2, 4, 8, 12, and 24 h postinjection. The blood samples were prepared by centrifugation for 10 min at 4500 rpm. The plasma, with a volume of 100 μL, was admixed with 100 μL of methanol by vortexing for 5 min, and then it was extracted with 1 mL of chloroform by vortexing for another 5 min. After centrifugation at 10000 rpm for 10 min at 4 °C, 800 μL of chloroform was collected and dried for further determination. The concentration of DOX was measured by HPLC. And the concentration of ADD was detected by HPLC using ultraviolet detector set at 300 nm. Water (0.1% trifluoroacetic acid) and acetonitrile, with HPLC grade, were chosen for the mobile phase. Pharmacokinetic parameters, including mean retention time (MRT), area under the curve (AUC), maximum drug concentration time (Tmax), half-life (t1/2), clearance (CL), and maximum drug concentration (Cmax), were calculated using the drug and statistics (DAS) software (version 2.1.1, Mathematical Pharmacology Professional Committee, China).

mitochondrial transmembrane potential assay kit according to the protocol of Beyotime Institute of Biotechnology. ATP Level. MCF7/ADR cells were treated with blank medium, DOX, ADD, DOX−ADD, TPGS, TPGS−NO, or DOX−ADD@TPGS−NO micelles, with DOX or NO concentration of 5 μM (the molar ratio of DOX to ADD was 1:1). After overnight incubation, cells were obtained, and the intracellular ATP level was detected by the ATP luminescence assay kit. 2.8. In Vivo Pharmacokinetics Study. Female adult Sprague-Dawley (SD) rats with a weight of 180−200 g were randomly divided into three groups. Free DOX, ADD, and DOX−ADD@TPGS−NO micelles were administered to SD rats intravenously at the dosage of 10 mg/kg DOX or 5.78 mg/ kg ADD (the molar ratio of DOX to ADD was 1:1), respectively (n = 4). Blood samples were drawn from the orbit of different formulations-treated rats with a glass capillary D

DOI: 10.1021/acs.molpharmaceut.7b00922 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 3. In vitro antitumor efficacy of DOX−ADD@TPGS−NO micelles in MCF7 and MCF7/ADR cells. Cell viability of (A−C) MCF7 and (D− F) MCF7/ADR cells after being treated with DOX, ADD, DOX−ADD, TPGS−NO, and DOX−ADD@TPGS−NO micelles at various concentrations for 24, 48, or 72 h, respectively.

Table 1. IC50 Values (μM) of DOX, ADD, DOX−ADD, TPGS−NO, and DOX−ADD@TPGS−NO micelles against MCF7 Cells and MCF/ADR Cells with Different Incubation Times incubation time

cells

DOX

ADD

DOX−ADD

TPGS−NO

DOX−ADD@TPGS−NO

24 h

MCF7 MCF7/ADR MCF7 MCF7/ADR MCF7 MCF7/ADR

16.10 >500 9.354 >500 8.57 20.47

10.49 13.14 6.41 11.95 5.35 8.79

13.71 28.70 12.72 19.88 4.49 16.81

15.90 22.67 7.88 19.20 5.76 12.64

7.95 12.48 6.68 7.79 5.43 5.80

48 h 72 h

2.9. Biodistribution. Tumor bearing mice with a tumor volume around 500 mm3 were intravenously injected with DIR@TPGS−NO micelles at DIR dosage of 2 mg/kg. For in vivo imaging, the mice were continuously imaged by a noninvasive optical imaging system (IVIS) (Pearl Trilogy, LICOR, USA) at the predetermined time points after administration. As for ex vivo observation of DIR@TPGS− NO biodistribution, mice were sacrificed, and tumors and major organs were collected at 1, 4, 8, 12, 24, 48, and 72 h postinjection. The fluorescence of DIR in each tissue was determined with the imaging system. The semiquantitative analysis of in vivo and ex vivo biodistribution in tumors and major organs was conducted by the living image software. 2.10. In Vivo Antitumor Effects of DOX−ADD@TPGS− NO Micelles. The antitumor efficacy of DOX−ADD@TPGS− NO micelles was investigated on a 4T1 tumor model. The 4T1 tumor model, with the characteristic of in situ metastasis, can serve as both a subcutaneous and metastatic model in the antitumor effects study. 4T1 cells, with a number of 1.0 × 105, were planted in situ at the right flank of the Balb/c mice. At day 6, the mice were divided into six groups (n = 7), when tumor volume was about 50−100 mm3. Different formulations, including saline, free-DOX, ADD, DOX−ADD, TPGS−NO, and DOX−ADD@TPGS−NO micelles at the dosage of 5 mg/ kg of DOX (the molar ratio of DOX to ADD was 1:1), were intravenously injected into the mice four times at day 6, 10, 14, and 18, respectively. Tumor growth and body weight were observed every 2 days. Three days after the last administration, the tumors and lungs were obtained, weighed, and imaged. For the metastatic observation, the lungs were fixed with Bovin fixative solution. Metastatic nodules on the lungs were counted. In addition, lungs were excised and stained with hematoxyline

and eosine (H&E) to observe the inner metastatic lung nodules. 2.11. In Vivo Safety Evaluation of DOX−ADD@TPGS− NO Micelles. For the safety assessment of DOX−ADD@ TPGS−NO micelles, different formulations, including saline, free-DOX, ADD, DOX−ADD, TPGS−NO, and DOX−ADD@ TPGS−NO micelles at the dosage of 5 mg/kg of DOX (the molar ratio of DOX to ADD was 1:1), were given to healthy mice. Blood samples were then collected to measure the hematological and biochemical parameters, including aspartate transaminase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN). Besides, the major organs were sectioned and fixed by 4% paraformaldehyde for H&E staining analysis. 2.12. Statistical Analysis. The results were shown as mean ± standard deviation (SD). Statistical analysis was conducted by one-way ANOVA with SPSS software (version 19.0). When *p < 0.05, it was considered to be a significant difference.

3. RESULTS 3.1. Preparation and Characterization of DOX−ADD@ TPGS−NO Micelles. The structures of the DOX−ADD conjugate and TPGS−NO are shown in Figure S1. DOX− ADD@TPGS−NO micelles were simply fabricated by a thinfilm hydration method. The micelles exhibited an average size of 10.0 ± 0.5 nm with homogeneous distribution. The surface morphology of DOX−ADD@TPGS−NO micelles was observed by TEM, which showed spherical shape (Figure 1A). The micelles exhibited good stability as there was no significant size change in pH 7.4 PBS and FBS for 7 days (Figure 1B). The drug encapsulation efficiency of DOX−ADD in the micelle E

DOI: 10.1021/acs.molpharmaceut.7b00922 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 4. Synergistic effects of ADD and TPGS for overcoming MDR in MCF7/ADR cells. (A) ROS generation in MCF7/ADR cells was measured and quantitatively analyzed by a flow cytometer. (B) Mitochondria membrane potential change in MCF7/ADR cells was detected by a flow cytometer. (C) Percentage of cells with decreased mitochondria membrane potential. (D) Intracellular ATP level in MCF7/ADR cells was measured under luminometer.

Figure 5. In vivo pharmacokinetics. Plasma concentration−time profiles of (A) DOX and (B) ADD in SD rats (n = 4) after intravenous administration of free-DOX, ADD, and DOX−ADD@TPGS−NO micelles at the dosage of 10 mg of DOX/kg or 5.78 mg of ADD/kg (the molar ratio of DOX to ADD was 1:1).

system was 90.3 ± 3.2%, which indicated less consumption of agents. As previously studied, TPGS−NO and DOX−ADD demonstrated redox and pH sensitive characteristics, respectively.17,24 The stimuli-response drug release behaviors of DOX−ADD@TPGS−NO micelles were consequently performed. NO release of DOX−ADD@TPGS−NO micelles mediated by redox environment was conducted in pH 7.4 PBS with or without 10 mM DTT. As shown in Figure S2A, the micelles demonstrated controlled and sustained release of NO under the redox condition. There was about 80% NO released

after 120 h. The linkage between DOX−ADD was a hydrazone bond with pH sensitivity. The release of DOX from micelles was, thus, performed at pH 5.0 ABS and pH 7.4 PBS by a dialysis method. The micelles demonstrated significant DOX release in pH 5.0 buffer compared with the ignorable DOX release in pH 7.4 PBS (Figure S2B). The results suggested that the breakage of the hydrazone bond in DOX−ADD indeed occurred at the acidic condition, and the micelles could achieve obvious pH-sensitive drug release. Owing to the same molar ratio of DOX and ADD in the conjugate of DOX−ADD, the release tendency of ADD would be in accordance with that of F

DOI: 10.1021/acs.molpharmaceut.7b00922 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Table 2. Pharmacokinetic Parameters of DOX and ADD after Intravenously Administration of DOX, ADD, and DOX−ADD@ TPGS−NO Micelles at the DOX Dosage of 10 mg/kg and ADD Dosage of 5.78 mg/kg parameters

unit

dose AUCa MRTb tl/2c Tmaxd CLe Cmaxf

mg/kg mg/L*h h h h L/h/kg mg/L

DOX 10 1.50 2.68 0.74 0.50 6.96 0.36

+ 0.36 + 2.84 ± 0.11 + 1.83 + 0.38

DOX−ADD@TPGS−NO (DOX) 10 23.11 3.70 3.22 0.50 0.44 10.75

± 3.15 + 0.65 ± 0.37 ± 0.06 ± 0.92

ADD 5.78 46.43 21.84 12.56 0.50 0.13 4.26

± 11.34 ± 6.27 ± 4.39 ± 0.034 ± 2.06

DOX−ADD@TPGS−NO (ADD) 5.78 79.25 ± 30.00 34.33 ± 9.51 33.22 ± 5.38 0.50 0.079 ± 0.025 18.65 ± 1.94

a

AUC: Area under the curve. bMRT: Mean retention time. ct1/2: Half-life dTmax: Maximum drug concentration time eCL: Clearance fCmax: Maximum drug concentration

Figure 6. In vivo and ex vivo biodistribution of DIR@TPGS−NO micelles in tumor-bearing mice. For in vivo distribution, the mice were continuously imaged. Tumors and major organs were collected for ex vivo observation at predetermined time intervals. (A) In vivo biodistribution. (B) Ex vivo biodistribution in tumors and major organs. (C) Semiquantitative analysis of ex vivo fluorescence in tumors and major organs.

DOX. The redox and pH dual-sensitive drug release characteristics of DOX−ADD@TPGS−NO micelles would be beneficial for the subsequent cancer treatment. 3.2. In Vitro Cellular Uptake of DOX−ADD@TPGS−NO Micelles. Efficient cellular uptake is the basic prerequisite for nanomedicine to exert cytotoxicity on tumor cells.27 Intracellular uptake of DOX−ADD@TPGS−NO micelles was conducted on MCF7 and MCF7/ADR cells by a flow cytometer. After incubation with different formulations, the cells were collected for DOX fluorescence detection. As seen from Figure 2A,B, the cellular uptake increased with the prolonged incubation time. DOX−ADD@TPGS−NO micelles exhibited significant higher cellular uptake in comparison to free-DOX and DOX−ADD in MCF7 cells. It may be attributed to that micelles could promote cellular uptake. A similar tendency was observed in MCF7/ADR cells. As a comparison with MCF7 cells, the reduced fluorescence in MCF7/ADR cells may be caused by the increased drug efflux due to the overexpressed P-gp in drug-resistant cells. At 4 h, the intracellular DOX fluorescence intensity of DOX−ADD@ TPGS−NO micelles in MCF7/ADR cells was 4.1-fold compared with that of free-DOX. DOX−ADD@TPGS−NO micelles exhibited the potential to overcome MDR, owing to the dual actions of mitochondria disruption and P-gp inhibition

by ADD and TPGS. For the direct visualization of cellular distribution, MCF7 and MCF7/ADR cells were observed under CLSM. After incubation for 4 h, the integral trend of DOX fluorescence intensity observed by confocal microscopy was inconsistent with the results detected by flow cytometry (Figure 2C,D). These results can infer that DOX−ADD@ TPGS−NO micelles could enhance in vitro cellular uptake both in drug-sensitive and -resistant cancer cells. 3.3. In Vitro Cytotoxicity of DOX−ADD@TPGS−NO Micelles. The enhanced cell uptake of micelles may induce increased cytotoxicity against MCF7 and MCF7/ADR cells. The cytotoxicity of different formulations was further performed on MCF7 and MCF7/ADR cells for 24, 48, or 72 h. The relative results are represented in Figure 3. DOX exhibited obvious cytotoxicity on MCF7 cells due to easy access to the nucleus. However, the cytotoxicity of DOX against MCF7/ADR cells was greatly limited and compromised. ADD, with the function of disrupting energy supply of mitochondria, was also found with obvious cytotoxicity on MCF7 and MCF7/ ADR cells. TPGS−NO micelles demonstrated moderate cell cytotoxicity on both MCF7 and MCF7/ADR cells. It may be ascribed to the tumor cell killing effects of released NO. Among all formulations, DOX−ADD@TPGS−NO micelles showed the most significant cytotoxicity on MCF7/ADR cells, which G

DOI: 10.1021/acs.molpharmaceut.7b00922 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 7. Investigation of in vivo antitumor efficiency of DOX−ADD@TPGS−NO micelles in a 4T1 subcutaneous tumor model. (A) Tumor volume of each mouse after being treated with saline, DOX, ADD, DOX−ADD, TPGS−NO, and DOX−ADD@TPGS−NO micelles (n = 7). (B) The overall tumor growth curve. (C) Tumor images and (D) tumor weight of different formulations at day 21 when the mice were sacrificed. (E) Body weight change during the treatment.

highest ROS level among all groups, which may be due to the mitochondria dysfunction effects of TPGS and ADD. Another indicator of mitochondria dysfunction is mitochondria membrane potential loss, which is ascribed to the change of integrity of mitochondrial membrane.29 In comparison to the control, DOX showed negligible decrease of membrane potential, while DOX−ADD@TPGS−NO micelles demonstrated obvious membrane potential loss which was indicated by the transfer of red fluorescence to green fluorescence (Figure 4B,C). ATP is closely related to the function of P-gp. The intracellular ATP level was further evaluated. In Figure 4D, free-ADD and TPGS micelles demonstrated downregulation of the ATP level, which was our expectation, and TPGS−NO micelles maintained the effect of TPGS. Relative low intracellular ATP level was observed in DOX−ADD@ TPGS−NO micelles, which was in accordance with the mitochondria membrane potential loss. As seen from above, DOX−ADD@TPGS−NO micelles showed great potential in

may be attributed to the direct cell killing effects of DOX and P-gp inhibition by TPGS and ADD. The relative IC50 values were calculated and are shown in Table 1. The resistance factor (RF) of DOX was over 31.06 at 24 h, while the RF of DOX− ADD@TPGS−NO micelles was 1.57, which indicated that DOX−ADD@TPGS−NO micelles showed great potential in overcoming MDR. 3.4. Synergistic Mechanisms of Overcoming MDR by TPGS and ADD. TPGS remains as an efficient inhibitor P-gp for overcoming MDR. The P-gp inhibition of TPGS was mainly attributed to mitochondria dysfunction, which was indicated by ROS generation, mitochondrial membrane potential loss, and ATP level reduction. 28 ADD was reported to disrupt mitochondria and decrease the ATP level, thus overcoming MDR.15 As shown in Figure 4A, enhanced ROS production was observed in free-DOX, ADD, DOX−ADD, TPGS, TPGS−NO, and DOX−ADD@TPGS−NO micelles compared with the control. DOX−ADD@TPGS−NO micelles exhibited the H

DOI: 10.1021/acs.molpharmaceut.7b00922 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Figure 8. Evaluation of the antimetastatic effects of DOX−ADD@TPGS−NO micelles in a 4T1 in situ metastatic tumor model. (A) Representative lungs images with metastatic nodules of different groups. (B) The number of metastatic nodules on lungs of different groups. (C) H&E staining of lungs for observation of inner nodules (bar 100 μm).

DOX, respectively. ADD was for the first time evaluated in pharmacokinetics study (Figure 5B). As seen from Table 2, the pharmacokinetic parameters of free-ADD, including AUC, MRT, t1/2, Cmax, and CL, are 35.24 ± 9.69 mg·h/L, 9.14 ± 0.99 h, 12.56 ± 4.39 h, 4.26 ± 2.06 mg/L, and 0.13 ± 0.034 L/h/kg, respectively. DOX−ADD@TPGS−NO micelles demonstrated improved pharmacokinetics profile of ADD. In detail, the AUC, t1/2, and Cmax of micelles were 1.40-, 2.64- and 4.38-fold compared with those of free-ADD, respectively. DOX−ADD@ TPGS−NO micelles enhanced drug concentration in plasma and exhibited prolonged circulation in body, which could be due to the protection effect of polyethylene glycol (PEG) shell in TPGS. 3.6. Biodistribution. Nanomedicine is supposed to accumulate at the tumor site as much as possible after circulation in body, as efficient drug accumulation in a tumor plays an important role in antitumor efficiency. In vivo biodistribution was further investigated by intravenously injecting DIR@TPGS−NO micelles to tumor bearing mice. Mice were imaged by IVIS at the predetermined intervals after administration. Semiquantitative analysis of the fluorescence

disturbing mitochondria by enhancing ROS generation, decreasing the mitochondria membrane potential and reducing the ATP level. The enhanced cytotoxicity on tumor cells may be attributed to the inhibition effects of P-gp by TPGS and ADD as well as the increased accumulation of DOX. 3.5. In vivo Pharmacokinetics Study. Long circulation of nanomedicine in blood could contribute to drug accumulation in tumor and, thus, improve antitumor efficiency. Pharmacokinetics study was conducted by intravenous injection of freeDOX, ADD, and DOX−ADD@TPGS−NO micelles at the dosage of 10 mg/kg of DOX or 5.78 mg/kg of ADD (the molar ratio of DOX to ADD was 1:1). Time-dependent drug concentration profiles of DOX and ADD are demonstrated in Figure 5A,B, respectively. Pharmacokinetic parameters were calculated and are summarized in Table 2. In comparison to the quick elimination of free-DOX, DOX−ADD@TPGS−NO micelles could maintain high concentration of dissociated DOX in plasma (Figure 5A). The AUC, MRT, t1/2, and Cmax of DOX for rats treated with micelles were 23.05 ± 3.11 mg·h/L, 2.79 ± 0.38 h, 3.22 ± 0.37 h, and 10.76 ± 0.92 mg/L, which were 16.60-, 1.40-, 4.35- and 29.47-fold higher than those of I

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Figure 9. Safety evaluation of DOX−ADD@TPGS−NO micelles. Blood physiochemical parameters, including (A) AST, (B) ALT, and (C) BUN, of mice after being administrated with saline, DOX, ADD, DOX−ADD, TPGS−NO, and DOX−ADD@TPGS−NO micelles. (D) H&E staining of hearts, livers, spleens, lungs, and kidneys (bar 100 μm).

TPGS−NO micelles was evaluated in a 4T1 subcutaneous and in situ metastatic tumor model. Tumor bearing mice were administrated four times with different formulations including saline, free-DOX, ADD, DOX−ADD, TPGS−NO, and DOX− ADD@TPGS−NO micelles at the equivalent dosage of 5 mg/ kg of DOX. Tumors and lungs were acquired 3 days after the last administration. Tumor volume of each mouse treated with different formulations was monitored and summarized in Figure 7A, which demonstrated good homogeneity. As seen from the results of the overall tumor growth in Figure 7B, ADD demonstrated negligible effects on tumor inhibition, and DOX−ADD showed moderate antitumor efficiency. FreeDOX and DOX−ADD@TPGS−NO micelles both exhibited significant inhibition on tumor growth. The similar tendency could also be seen from the results of tumor images in Figure 7C and the tumor weight in Figure 7D. DOX is an acknowledged broad spectrum antitumor drug, which can exert great antitumor effects. The compromised antitumor effect of ADD may be ascribed to the relative low dosage in the treatment as well as the restricted effect on mitochondria.

intensity in tumor was conducted by a living image software. As shown in Figures 6A, S3, and S4A, the fluorescence in tumors increased with time going on and was maintained at high level from 4 to 24 h. For more direct observation of micelles distribution, tumors and major organs were imaged at 1, 4, 8, 12, 24, 48, and 72 h (Figure 6B). The ex vivo fluorescence in tumors and major organs was also semiquantitatively analyzed, which is shown in Figures S4B and 6C. The micelles were primarily distributed at livers at early time, while obviously enhanced fluorescence was observed in tumors from 4 h and the strongest intensity appeared at 8 h. The fluorescence at the tumor site was the highest among all organs from 8 to 72 h. The results of in vivo and ex vivo biodistribution demonstrated that the micelles exhibited significant accumulation at tumor site and even maintained high concentration for a long period of time. It may have benefited from the small size of micelles, protection of PEG shell in TPGS, and enhanced accumulation by released NO. 3.7. In Vivo Antitumor Effects of DOX−ADD@TPGS− NO Micelles. In vivo antitumor efficiency of DOX−ADD@ J

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ROS generation, decreased mitochondria membrane potential, and downregulated intracellular ATP level by the mitochondria dysfunction effects of TPGS and ADD. Moreover, significantly enhanced accumulation of micelles at the tumor site was observed in the biodistribution study. The micelles further demonstrated obvious tumor inhibition and antimetastatic efficiency in breast cancer. This work provides a good option for nanomedicine design based on the “molecular economy” principle and clinical application in the future.

DOX−ADD@TPGS−NO micelles demonstrated obvious tumor inhibition, which can be explained by the direct tumor killing effects of DOX, dual suppression on mitochondria by TPGS and ADD, and enhanced drug delivery by the released and diffused NO in vessels. Owing to the fact that the dosage ratio of different agents was not under investigation, the antitumor efficacy was compromised, and relative work should be further explored. Moreover, body weight of mice in all groups was in the normal range (Figure 7E), indicating good safety of the micelle system. To evaluate the effect on metastasis inhibition, lungs, as major organs for metastasis of breast cancer, were obtained and are presented in Figure S5. The sectioned lungs were further fixed in Bovin fixative solution overnight with metastatic nodules counted. Lungs with metastatic nodules are shown in Figure 8A, in which the representative nodules are pointed out by blank arrows. The number of nodules in different groups was summarized, which was 47 ± 12, 33 ± 5, 32 ± 6, 28 ± 3, 31 ± 6, and 19 ± 3 for saline, free-DOX, ADD, DOX−ADD, TPGS−NO, and DOX−ADD@TPGS−NO micelles, respectively. The number of nodules in saline was 2.5-fold compared with that of DOX−ADD@TPGS−NO micelles. As shown in Figure 8B, free-DOX, ADD, DOX−ADD, and TPGS−NO micelles showed moderate effects on metastasis inhibition in comparison with saline, while DOX−ADD@TPGS−NO micelles exhibited the best antimetastatic efficiency among all of the groups. Although ADD and TPGS−NO micelles showed negligible antitumor effects in the subcutaneous tumor, moderate antimetastasis efficiency was observed in the in situ metastasis model. To observe the inner metastatic nodules, lungs were excised and stained with H&E. Inner metastatic nodules were indicated by the orange dotted line in Figure 8C. In comparison to saline with obvious metastasis, there was hardly any metastasis observed in DOX−ADD@TPGS−NO micelles. In summary, DOX−ADD@TPGS−NO micelles demonstrated great potential in metastasis inhibition of breast cancer. 3.8. Safety Evaluation of DOX−ADD@TPGS−NO Micelles. Safety concern of nanomedicine is essential in cancer treatment. Therefore, a safety evaluation of DOX− ADD@TPGS−NO micelles was conducted. After administration of various formulations, blood samples and major organs were collected. The blood biochemical parameters, including AST, ALT, and BUN, were measured. Liver and kidney are the two major metabolic organs, and levels of AST and ALT in plasma are important indicators of the physiological condition of liver. The abnormal BUN level in blood can represent the damage of a kidney. In all groups, the AST, ALT, and BUN levels were in normal range, indicating good safety of the formulations (Figure 9A, B, and C). Major organs were sectioned, fixed, and stained with H&E. As seen from Figure 9D, there were no obvious pathological sites observed in all of the organs of different groups, which further evidenced the safety of DOX−ADD@TPGS−NO micelles.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00922. Structures of DOX−ADD conjugate and TPGS-based NO donor (TPGS−NO); cumulative NO release of micelles with or without 10 mM DTT (n = 3) and cumulative DOX-release of micelles at pH 5.0 and pH 7.4, respectively (n = 3); in vivo biodistribution of DIR@ TPGS−NO micelles at tumor site (n = 3); semiquantitative analysis of biodistribution of DIR@TPGS− NO micelles at tumor site; and images of fresh lungs in a 4T1 in situ metastatic tumor model (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Fax and Phone: +86-27-83601832; E-mail: zhipingzhang@ mail.hust.edu.cn. ORCID

Zhiping Zhang: 0000-0002-9235-5321 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (81373360 and 81673374), the Fundamental Research Funds for the Central Universities (2015ZDTD048), and the Applied Fundamental Research Program of Wuhan (2017060201010146). It was also supported by the Opening Project of Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education (Sichuan University). We thank the Analysis and Testing Center of Huazhong University of Science and Technology for TEM measurements.

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REFERENCES

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4. CONCLUSION In summary, a micelle system based on the “molecular economy” principle was constructed here to improve drug delivery, overcome MDR, and inhibit metastasis of breast cancer. DOX−ADD@TPGS−NO micelles made the utmost use of each component and exerted good antitumor efficiency. The micelles exhibited enhanced cell uptake and cytotoxicity on MCF7/ADR cells. The related mechanisms involved promoted K

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DOI: 10.1021/acs.molpharmaceut.7b00922 Mol. Pharmaceutics XXXX, XXX, XXX−XXX