Paclitaxel and Tacrolimus Coencapsulated Polymeric Micelles That

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Paclitaxel and tacrolimus co-encapsulated polymer-ic micelles enhance therapeutic effect of drug-resistant ovarian cancer Ning Wang, Tao He, Yangmei Shen, Linjiang Song, Ling Li, Xi Yang, Xia Li, Mengru Pang, Weijun Su, Xinyu Liu, Qinjie Wu, and Changyang Gong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09340 • Publication Date (Web): 26 Jan 2016 Downloaded from http://pubs.acs.org on January 29, 2016

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ACS Applied Materials & Interfaces

Paclitaxel and tacrolimus co-encapsulated polymeric micelles enhance therapeutic effect of drugresistant ovarian cancer Ning Wang

1,‡

, Tao He

1, ‡ ,

Yangmei Shen

2, ‡ ,

Linjiang Song 1, Ling Li 1, Xi Yang 1, Xia Li 1,

Mengru Pang 1, Weijun Su 3, Xinyu Liu 1,*, Qinjie Wu 1,*, Changyang Gong 1,* 1

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan Univer-

sity, and Collaborative Innovation Center for Biotherapy, Chengdu, 610041, P. R. China 2

Department of Gynecology and Obstetrics, Second West China Hospital, Sichuan University,

Chengdu, 610041, P. R. China 3

School of Medicine, Nankai University, Tianjin, 300071, P. R. China

*

To whom should be corresponded (C Gong, Q Wu, and L Liu). Tel: 86-28-85164063, Fax: 86-

28-85164060, E-mail: [email protected], [email protected], [email protected] ‡These

authors contributed equally.

ACS Paragon Plus Environment

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ABSTRACT The combination of chemotherapy drugs and multidrug-resistant (MDR)-reversing agents for treating MDR in tumors has attracted increasing attention. However, the poor water solubility of some anticancer drugs restricted their clinical application. In this work, we prepared poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) micelles as co-delivery system to load chemotherapy drug paclitaxel (PTX) and MDR-reversing agent tacrolimus (FK506). The PTX and FK506 co-loaded MPEG-PCL micelles (P-F/M) were prepared by a one-step solid dispersion method without any surfactants, toxic organic solvent or severe experimental conditions. The P-F/M had small particle size (28.7 ± 3.2 nm) and high encapsulation efficiency (99.3 ± 0.5%). Compared with A2780s cells (PTX-sensitive human ovarian cancer cells), the P-F/M showed a stronger cytotoxicity and an improving intracellular drug concentration of PTX than PTX loaded micelles (PTX/M) in A2780/T cells (PTX-resistant human ovarian cancer cells). Furthermore, P-F/M co-delivery system showed a more significant G2/M arrest and apoptosis induction effects, as well as activating apoptosis protein signaling pathway in A2780/T cells than those in A2780s cells. In summary, the results suggested that the co-delivery micelles of PTX and FK506 may serve as a potential candidate against MDR human ovarian cancer.

KEYWORDS: multidrug-resistant; ovarian cancer; micelles; co-delivery; Paclitaxel; Tacrolimus


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1. INTRODUCTION Ovarian cancer is the second most common cancer and the leading cause of death from cancer among women in the world, and it has been a serious threat to women's lives 1. Current ovarian cancer therapy mainly composed of chemotherapy in different stages of patients, including paclitaxel (PTX), docetaxel, gemcitabine, doxil, platinum compounds 2. However, the pathophysiological phenomenon which tumor cells become simultaneously resistant to different drugs after exposure to a single chemotherapeutic drug, a trait as multidrug resistance (MDR), is a major clinical obstacle to chemotherapy 2. MDR is mainly due to the overexpression of efflux transporters such as P-glycoprotein (P-gp)

3, 4

, which acts a key role in pumping many drugs out of

cancer cells. This process can make the intracellular chemotherapy concentrations below the effective value 5, and the anti-tumor therapeutic effects are three times less than those do not express P-gp 6. PTX, a natural extractive compound from the Pacific yew tree (Taxus brevifolia), is effective in treating a variety of solid tumors, such as ovarian cancer, breast cancer, lung cancer, etc 7. However, in ovarian cancer for example, with the application of PTX, more than 70% of initially diagnosed patients are resistant to PTX therapy, and eventually all of them will resistant upon relapse 8, 9. In the past few decades, the researchers have tried to find agents against the MDR of cancer cells, such as verapamil, quinidine, progesterone, tamoxifen, phenothiazines, and cyclosporine A

10

. Tacrolimus (FK506), which has been commonly used as immunosuppressants in

organ transplants

11, 12

, was found to be an effective inhibitor of P-gp 13. Due to suppressing the

drug exclusion of P-gp, FK506 has a profound potential role in reversing MDR 14. However, high inherent side effects and alternative of the pharmacokinetics of anticancer drugs, these MDRreversing agents had limited to clinical application 15. Some studies have found that the combina-


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tion of MDR-reversing agents and chemotherapy drugs could overcome drug resistance in cancer 4, 15, 16, 17.

Over the past few decades, biocompatible micelles, as delivery vehicles for chemotherapeutic agents, have attracted more attentions. Some researchers have shown that these nanocarriers can significantly enhance anti-tumor efficiency of various chemotherapeutic drugs with fewer side effects

18

. In addition, micelles can make the combination of chemical sensitization agents

and the chemotherapy drugs come true 19. The polymeric micelles were widely used as drug delivery system (DDS) with their core-shell morphology

20

, and our previous study have proved

that the preparation methods of poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) micelles were simple and did not need severe conditions, toxic organic solvent and surfactant, which make the micelles economic, low toxicity, and low side effects 21. In order to achieve the treatment of the combination of FK506 with PTX, the hydrophobic core of micelles was designed, which was also the key to many other hydrophobic drugs, such as curcumin and honokiol 22, 23, 24

.

In this study, PTX loaded MPEG-PCL micelles (PTX/M), FK506 loaded MPEG-PCL micelles (FK506/M) and PTX-FK506 dual-drugs combination micelles (P-F/M) were prepared and characterized. The release behavior of the co-delivery system was investigated. We also studied the intracellular drug concentration of P-F/M on A2780/T cells in vitro. We conducted tests of cell viability, cell cycle arrest and apoptosis on A2780/T and A2780s cells to compare the anticancer effect of P-F/M. The P-F/M co-delivery system may serve as a potential candidate for reversing MDR treatment.

2. MATERIALS AND METHODS


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2.1 Materials and cell lines Poly(ethylene glycol) methyl ether (MPEG, Mn=2000, Fluka, USA), ε-caprolactone (ε-CL, Alfa Aesar, USA), Stannous octoate (Sn(Oct)2, Sigma, USA), acetonitrile (HPLC grade, Sigma, USA), methyl thiazolyl tetrazolium (MTT, Sigma, USA), Tacrolimus (FK506, >98% by HPLC, Shanghai PureOne Biotechnology, Shanghai, China), paclitaxel (PTX; >98% by HPLC, Shanxi Senfu Biotechnology, Shanxi, China), propidium iodide (PI, Sigma, USA), Annexin V-FITC Apoptosis Detection Kit (KeyGEN Biotech, Nanjing, China), antigens for Western Blots (Abcam, USA) were used as received. All the organic solvents (acetone, dichloromethane, and diethyl ether) used in this article were analytic reagent (AR grade) and used as received. The paclitaxel-sensitive human ovarian cancer cell lines (A2780s) or paclitaxel-resistant human ovarian cancer cell lines (A2780/T) came from the State Key Laboratory of Biotherapy, Sichuan University, and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, USA) or Roswell Park Memorial Institute 1640 medium (RPMI 1640, Gibco, USA) with 10% fetal bovine serum (FBS, Caoyuan lvye, Huhht, China), respectively. For A2780/T cell culture, 80 µg/mL of PTX was added. The cells were grown in a 37oC incubator with a humidified 5% CO2 atmosphere. 2.2 Synthesis of the MPEG-PCL copolymer The MPEG-PCL diblock copolymer was prepared by ring-opening polymerization of ε-CL initiated by MPEG, and Sn(Oct)2 was used as the catalyst, which was described in our previous contributions in detail 25. Briefly, the calculated amount of MPEG and ε-CL were introduced into a dry glass ampoule under a nitrogen atmosphere, and a calculated amount of Sn(Oct)2 was added into the reaction vessel under mild magnetic stirring. The reaction system was kept at 130oC for 6 hours. The final products were dissolved in dichloromethane and reprecipitated from filtrate


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using diethyl ether. Then MPEG-PCL copolymer was vacuum dried to constant weight, dialyzed against water 4-6 times for 2 days, and then lyophilized and stored in glass desiccator or at -20oC for further use. Molecular weight of synthesized MPEG-PCL copolymer was 3800 (2000-1950), calculated by 1H-NMR. 2.3 Preparation and characterization of drug-loaded micelles The P-F/M were prepared by a one-step solid dispersion method 7, 26. Briefly, 2 mg of PTX, 2 mg of FK506 and 40 mg of MPEG-PCL copolymer were co-dissolved in 2ml acetone. Then, the solution was evaporated in a rotary evaporator at 60oC. During this process, homogenous thin film was obtained, and PTX and FK506 were encapsulated in MPEG-PCL copolymer as an amorphous substance. Then, the thin film was dissolved in water at 60oC to self-assemble into micelles. The P-F/M solution was filtered through a 0.22 µm syringe filter (Millex-LG, Millipore Co., USA) to remove the non-entrapped drug. The FK506/M and PTX/M were prepared by the same method. The particle size distribution, polydisperse index (PDI) and zeta potential of the blank micelles and drug-loaded micelles water solution were determined by Malvern Nano-ZS 90 laser particle size analyzer at room temperature. The morphological characteristics were observed by transmission electron microscopy (TEM, H-6009IV, Hitachi, Japan)

27

.

The PTX powder, FK506 powder, mixture of PTX, FK506 and MPEG-PCL (with the same ratio in P-F/M), and P-F/M were carried out crystallographic assay by x-ray diffractometer (X’Pert Pro, Philips, Eindhoven, Netherlands) using Mo Kα radiation. The concentration of PTX or FK506 loaded in micelles was determined by a high performance liquid chromatography instrument (HPLC, Waters, Japan). The drug loading (DL) and encapsulation efficiency (EE) of the PTX/M, FK506/M and P-F/M were determined according to


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formula eqn (1) and (2) 28. Briefly, 20 mg of lyophilized drug loaded micelles were dissolved in 0.2 mL of acetonitrile. The amount of PTX or FK506 in the solution was determined by HPLC. The DL and EE of the micelles were calculated according to formula (1) and (2): DL= Drug / (Polymer + drug) ×100%

(1)

EE= Experimental drug loading / Theoretical drug loading × 100%

(2)

2.4 Drug release in vitro The in vitro release behaviors of PTX and FK506 from micelles were studied by a modified dialysis method as previously described 29. Free PTX or FK506 was dissolved in DMSO-water solution, and the concentration of PTX or FK506 was 1 mg/mL that was the same dose as PTX/M, FK506/M and P-F/M. The solutions were placed in dialysis bags (molecular mass cutoff is 3.5kDa). The dialysis bags were incubated in 5 mL of PBS (pre-warmed to 37oC, pH 7.4) containing Tween80 (0.5%wt) or 5mL PBS (pre-warmed to 37oC, pH 7.4) containing 10% FBS at 37oC with gentle shaking (100 rpm) in 15 mL BD tube. At specific time points, 1mL release media was collected, and the remaining media was replaced by pre-warmed fresh release media. After being centrifuged, the supernatants were collected and stored at -20oC until analyzing by HPLC. 2.5 In vitro cytotoxicity analysis MTT method was used to test the cytotoxicity of PTX and FK506 as well as their combination against A2780s cells and A2780/T cells. Briefly, The A2780s cells and A2780/T cells were plated in 96-well plates and grown over night. Then the cells were exposed to a series of different concentrations of PTX/M (0-50 µg/mL on A2780s cells, 0-500 µg/mL on A2780/T cells), and four specific concentrations of FK506/M (2 µg/mL, 4 µg/mL, 8 µg/mL, 16 µg/mL, and 32 µg/mL) for 48 hours, respectively. Untreated cells were used as controls. The viability of the


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cells was measured using MTT method. In addition, the blank micelles and FK506/M were used as control. 2.6 Cell cycle analysis The induction of cell cycle arrest of the micelles was observed using the detection of chromosomal DNA stained with PI. A2780s and A2780/T cells were exposed to medium containing blank micelles, FK506/M, PTX/M (2 µg/mL on A2780s cells, 44 µg/mL on A2780/T cells) or PF/M (2 µg/mL PTX and 16 µg/mL FK506 co-loaded micelles on A2780s cells, 44 µg/mL PTX and 16 µg/mL FK506 co-loaded micelles on A2780/T cells) for 24 hours, respectively (based on the IC50 value calculated from MTT test for A2780s or A2780T cells). After washing with PBS, the cells were fixed with pre-chilled ethanol for 30 min at room temperature, then stained with PI (50 µg/mL in PBS) for 15 min. Flow Cytometry (BD FACS, Calibur, BD, USA) was used to analyze cycle arrest. 2.7 In vitro apoptosis detection Apoptosis assay was evaluated by FCM (BD FACS Calibur, BD, USA) analysis using FITC-conjugated Annexin V/PI (KeyGen, Nanjing, China) staining as the manufacturer's instructions. Briefly, A2780s and A2780/T cells were incubated in 6-well plates for 12 hours, and exposed to medium containing blank micelles, FK506/M, PTX/M (2 µg/mL on A2780s cells, 44 µg/mL on A2780/T cells) or P-F/M (2 µg/mL PTX and 16 µg/mL FK506 co-loaded micelles on A2780s cells, 44 µg/mL PTX and 16 µg/mL FK506 co-loaded micelles on A2780/T cells) for 24 hours, respectively (based on the IC50 value calculated from MTT test for A2780s or A2780T cells). Flow Cytometry (BD FACS, Calibur, BD, USA) was taken advantages to detect apoptosis. Both early apoptotic (AnnexinV-positive, PI-negative) and late apoptotic (AnnexinV-positive and PI-positive) cells were included in cell apoptosis determinations.


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2.8 Intracellular drug concentration of PTX The intracellular drug concentration of PTX was quantitated in A2780s cells and A2780/T cells, respectively. The A2780s cells and A2780/T cells were cultured in 6-well plates and grew for 12 hours. Then the growth medium was removed and the cells were washed with PBS for twice, and a serum-free medium contained a certain amount of PTX/M (2 µg/mL on A2780s cells, 44 µg/mL on A2780/T cells) or P-F/M (2 µg/mL PTX and 16 µg/mL FK506 co-loaded micelles on A2780s cells (based on the IC50 value calculated from MTT test for A2780s or A2780T cells), 44 µg/mL PTX and 16 µg/mL FK506 co-loaded micelles on A2780/T cells) were given to A2780s cells and A2780/T cells, respectively. After incubation for 0, 2, and 4 hours, the cells were collected and lysed with RIPA (Sigma) for 30 min on ice. PTX was extracted by a mixture of ethyl acetate and n-hexane (1 : 3). After dried by nitrogen, the samples reconstituted in acetonitrile were measured by HPLC (Waters, Japan). 2.9 Western bolt The A2780s cells and A2780/T cells were cultured at dishes and grown for 12 hours. Then the two kinds of cells were treated with blank micelles, FK506/M, PTX/M (2 µg/mL on A2780s cells, 44 µg/mL on A2780/T cells) or P-F/M (2 µg/mL PTX and 16 µg/mL FK506 co-loaded micelles on A2780s cells, 44 µg/mL PTX and 16 µg/mL FK506 co-loaded micelles on A2780/T cells) (based on the IC50 value calculated from MTT test for A2780s or A2780T cells). After incubated for 24 hours, the A2780s cells and A2780/T cells were collected. The cells precipitation was dealt by buffer containing 1% triton X-100, 1% deoxycholate, and proteinase inhibitor cocktail (Sigma) for 30 min on ice, After centrifuged for 15 min at 4oC, the supernatant was collected. The protein supernatant was stored at -80oC before used. The samples were detected by 12% SDS-PAGE and incubated with antibodies (Abcam) including Caspase 3, Cleaved Caspase 3,


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Caspase 8, Cleaved Caspase 8, Caspase 9 or Cleaved Caspase 9, respectively. Blots were developed with horseradish peroxidase (HRP)-conjugated secondary antibodies and chemiluminescent substrate on Kodak X-ray films. 2.10 STATISTICAL ANALYSIS The statistical analysis was carried out using SPSS 19.0 software (Chicago, IL, USA). P value

Paclitaxel and Tacrolimus Coencapsulated Polymeric Micelles That

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