Article pubs.acs.org/Biomac
Oral Nanomedicine Based on Multicomponent Microemulsions for Drug-Resistant Breast Cancer Treatment Ding Qu,†,‡ Lixiang Wang,† Meng Liu,§ Shiyang Shen,§ Teng Li,§ Yuping Liu,†,‡ Mengmeng Huang,†,‡ Congyan Liu,†,‡ Yan Chen,*,†,‡ and Ran Mo*,§ †
Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210028, China ‡ Jiangsu Provincial Academy of Traditional Chinese Medicine, Nanjing 210028, China § State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center of Advanced Pharmaceuticals and Biomaterials and Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, China S Supporting Information *
ABSTRACT: The aim of this study is to demonstrate the enhanced therapeutic efficacy of anticancer drugs on drug-resistant breast cancer using multicomponent microemulsions (ECG-MEs) as an oral delivery system. The etoposide-loaded ECG-MEs were composed of coix seed oil and ginsenoside Rh2 (G-Rh2), both of which possess not only the synergistic antitumor effect with etoposide, but also have excipient-like properties. Orally administrated ECG-MEs were demonstrated to be able to accumulate at the tumor site following crossing the intestines as intact vehicles into the blood circulation. The spatiotemporal controlled release characteristics of ECG-MEs brought about the efficient P-gp inhibition by the initially released G-Rh2 and the increased intracellular accumulation of the sequentially released etoposide. The combination antitumor activity of etoposide, GRh2 and coix seed oil using ECG-MEs was verified on the xenograft drug-resistant breast tumor mouse models. In addition, the safety evaluation studies indicated that treatment with ECG-MEs did not cause any significant toxicity in vivo. These findings suggest that ECG-MEs as an oral formulation may offer a promising strategy to treat the drug-resistant breast cancer.
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INTRODUCTION Breast cancer is the most commonly diagnosed cancer (15% of all new cancers) in China and the leading cause of cancer death in Chinese women younger than 45 years.1 Surgery resection, radiation therapy and chemotherapy, Traditional Chinese Medicine (TCM), and palliative care are currently recommended as standard treatments for breast cancer in China.2 Thereinto, adjuvant chemotherapy is a common regimen in China, as one report noted that 81.4% of Chinese patients with invasive breast cancer had experience of adjuvant chemotherapy.3 Unfortunately, many treated patients have to abort the therapy in advance (less than the minimum recommended standard) due to inevitable multidrug resistance (MDR) induced by various chemotherapeutic agents with unrelated structures or mechanisms.4 The MDR-related desensitization of tumor cells to anticancer drug led to an unsuccessful treatment for over 90% of patients after a couple of courses of chemotherapy. 5 Several mechanisms contribute to the occurrence of MDR, including increased efflux transporter, decreased drug influx, endolysosomal drug entrapment, © XXXX American Chemical Society
activation of antiapoptotic pathways, and alteration of cell cycle regulation.6−8 As one of the most consistently overexpressing transport proteins on the MDR tumor cell membrane, P-glycoprotein (P-gp) has attracted considerable attention.9 Over the past decade, coadministration of chemotherapeutic agents with specific P-gp inhibitors that can interfere with the function of P-gp, such as verapamil and cyclosporine A, results in an apparent enhancement on the MDR cellular uptake in vitro.10 Oral administration is a highly preferred approach for patients due to the acceptable satisfaction and compliance,11−13 which brings about increasing attention to overcome MDR through integration of functional agents and anticancer drugs into one delivery system.14−16 A nanosized delivery system coencapsulating P-gp inhibitors and anticancer drugs offers a promising strategy for overcoming MDR by the inherently Received: January 3, 2017 Revised: March 4, 2017
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DOI: 10.1021/acs.biomac.7b00011 Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 1. Schematic design of multicomponent microemulsions, ECG-MEs, for oral delivery of anticancer drug to overcome the MDR tumor.
property, we believe that multifunctional G-Rh2 might have significant contribution to achieving stronger anti-MDR effect, superior combination therapy and lower systematic toxicity after incorporation into EC-MEs. Forasmuch as the advantage of such strategy, we are highly interested in the potential of orally administrated multicomponent microemulsion for overcoming MDR and enhancing the antitumor efficacy. Herein, we report multicomponent microemulsions (ECGMEs) consisting of etoposide (chemotherapeutic agent), coix seed oil (oil phase, anticancer component), G-Rh2 (P-gp inhibitor, mimic surfactant, and anticancer component), RH40 (surfactant), and PEG400 (cosurfactant) for oral delivery of etoposide to treat the MDR tumor (Figure 1). Orally administrated ECG-MEs can inhibit the drug efflux of P-gp overexpressed on the enterocytes and efficiently cross the intestinal barrier as intact vehicles. After oral absorption, ECGMEs are able to accumulate at the tumor site by the EPR effect. Surfactant-like G-Rh2 localized in the outer layer of ECG-MEs is released much faster than etoposide encapsulated within the oil phase. At the tumor tissue, the initially released G-Rh2 suppresses P-gp, which facilitates the increased diffusion of the extracellularly released etoposide into the MDR cells and also the enhanced retention of the intracellularly released etoposide within the MDR cells. In the MDR tumor cells, the released etoposide, G-Rh2, and coix seed oil produce the synergistic antitumor effects.
enhanced intestinal retention/absorption and capability of escaping from P-gp-mediated efflux.17 However, few formulations have been successfully exploited until now, since several big challenges to control the release sequence and the in vivo structural integrity remain ambiguous, which are of great importance to repairing the abnormal P-gp expression of the MDR tumor microenvironment, and realizing desirable tumor accumulation by the enhanced permeability and retention (EPR) effect, respectively. In addition, several P-gp inhibitors including verapamil, cyclosporine A, Cremophor EL, and Pluronic copolymer have drawbacks of an insufficient amount of release,10 immunologic suppression,18 and hypersensitivity for affecting patients compliance.12 To this end, it is highly desirable to pursue a specific P-gp inhibitor that possesses excipient-like properties and anticancer effect simultaneously, which is favorable for realizing the controllable release, stable structure, and synergistic antitumor treatment. Ginsenoside Rh2 (G-Rh2) that was first isolated from the red ginseng as a major effective component of ginsenosides has been widely used as a promising therapeutic tool to regulate immunologic function, induce cell apoptosis, and inhibit cell growth inhibition on a variety of tumor types, including colorectal,19 breast,20 and liver cancers.21 Nevertheless, most previous studies focused on the application of G-Rh2 for combination cancer therapy due to its low side effect and high anticancer efficacy. G-Rh2 also has a remarkable ability to enhance the uptake of the MDR tumor cells via interacting with the overexpressing P-gp.22 More significantly, the chemical structure of G-Rh2 containing a hydrophilic group, dextrose, and a hydrophobic segment, steroid, which endues G-Rh2 with amphiphilicity for fabricating the nanocarriers as a “pharmacologically active” material. Etoposide is a well-known potent anticancer drug, but prone to the occurrence of MDR and severe systemic side effects.23 Until now, its commercial oral formulation is incapable of addressing drug efflux, although improving the intestinal absorption.24 In our previous study,25 we have developed etoposide-loaded coix seed oil microemulsion (EC-MEs) by replacing conventional oil excipient with coix seed oil, which has been approved by the Chinese Ministry of Public Health for application as an antineoplastic therapy. The obtained EC-MEs presented great effects on enhancing the cell apoptosis induction and improving antitumor activity. Inspired by the fact that coix seed oil with both anticancer effect and oil phase
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EXPERIMENTAL SECTION
Materials. Coix seed oil was prepared by supercritical CO2 extraction technology (purity > 85%, determined by ultraviolet spectroscopy using glyceryl trioleate as a reference substance). GRh2 was purchased from Aladdin Industrial Co. (Shanghai, China). PEG400, rhodamine 123 (R123), and etoposide were purchased from Sigma-Aldrich Co., Ltd. (Poole, U.K.). Cremophor RH40 was provided by BASF Co., Ltd. (Ludwigshafen, Germany). Verapamil, amiloride, sodium azide, genistein, sucrose, and ammonium chloride were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Other chemicals and solvents were analytical grade. Preparation and Characterization. ECG-MEs were synthesized through the similar preparation method of EC-MEs, as previously described, but with some modifications.25 In brief, etoposide (50 mg) was added into coix seed oil (4.0 g) with stirring for 2 h. Cremophor RH40 (3.7 g), G-Rh2 (0.3 g), and PEG400 (1.0 g) were mixed thoroughly. Afterward, the mixed surfactant was slowly added into the etoposide oil solution with further vigorous mechanical stirring at 2000 B
DOI: 10.1021/acs.biomac.7b00011 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules rpm using the LCD Digital Enhanced Overhead Stirrer (OS40-Pro, SCILOGEX) at 4 °C until the mixture became completely homogeneous. The final ECG-MEs formulation was obtained after dropwise adding 20.0 mL of deionized water into the above mixture. EC-MEs without the G-Rh2 component as a control were prepared through assembly of adlay seed oil (4.0 g), Cremophor RH40 (4.0 g), and PEG400 (1.0 g) in the aqueous phase. The average hydrodynamic diameter, polydispersity index (PDI), and zeta potential of ECG-MEs were determined using the dynamic light scattering (DLS) measurement (Nano ZS, Malvern). The morphology of ECG-MEs was examined using transmission electron microscope (TEM). Briefly, the freshly prepared ECG-MEs were first suspended in phosphate buffer saline (PBS) and then dropped onto a film-coated copper grid, followed by staining with a drop of 1% (wt %) phosphotungstic acid. After air-drying, the sample was observed using TEM (JEM-200CX, JEOL). The contents of etoposide and G-Rh2 in ECG-MEs were quantified using HPLC (1260 Infinity system, Agilent) with a reverse phase C18column (4.6 mm × 150 mm × 5 μm; Diamond). The column temperature was set at 30 °C and the flow rate was set at 1 mL/min. For the etoposide quantification, the mobile phase was the mixture of methanol and water (55:45, v/v) and the detection wavelength was set at 285 nm. For the G-Rh2 quantification, the mobile phase was the mixture of methanol, acetonitrile, and water (8:8:1, v/v/v) and the detection wavelength was set at 203 nm. The encapsulation efficiency (EE) of etoposide or G-Rh2 was calculated as EE (%) = C × V/W × 100%, where C is the concentration of etoposide or G-Rh2 in the obtained microemulsion; V is the volume of the microemulsion; and W is the feeding weight of etoposide or G-Rh2. In Vitro Drug Release. The release property of etoposide and GRh2 from ECG-MEs was determined using the dialysis method. A total of 5 mL of ECG-MEs containing 5 mg etoposide and 30 mg G-Rh2 was added into a dialysis bag (MWCO 10 kDa) and then immersed in 500 mL of phosphate buffer (PB; pH 7.4), artificial intestinal fluid, and gastric fluid containing 1% (w/v) Tween 80 at 37 °C under stirring at 60 rpm, respectively. At predetermined time intervals, 1 mL of buffer solution was sampled and filtered through a polycarbonate membrane filter (0.22 μm pore size), followed by supplementing 1 mL of corresponding fresh medium. The quantities of etoposide and G-Rh2 released from ECG-MEs were determined using HPLC. Cell Culture. The human breast carcinoma cells (MCF-7), human colon adenocarcinoma cells (Caco-2), and multidrug-resistant MCF-7 (MCF-7/MDR) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% (v/v) fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 μg/mL) under an atmosphere of 5% CO2 and 90% relative humidity in an incubator (Thermo 3111). Cellular Uptake and Intracellular Delivery. To visualize intracellular transport, R123, a P-gp substrate with green fluorescence,12,26 was used to substitute etoposide and encapsulated in the GRh2-containing coix seed oil microemulsion (CG-MEs) with a feeding ratio of R123 to coix seed oil of 1/40000 (w/w) to obtain R123loaded CG-MEs (R123/CG-MEs). Other R123 formulations, including free R123, the mixture of R123 and G-Rh2 (or verapamil), R123-loaded coix seed oil microemulsion without the G-Rh2 component (R123/C-MEs), and the mixture of R123/CG-MEs and G-Rh2 were taken as control groups to validate the effect of ECG-MEs on enhancing intracellular accumulation in MCF-7/MDR cells. Three different cells, Caco-2, MCF-7/MDR, and MCF-7 cells, were seeded in six-well plates at a density of 1 × 105 cells/well. After reaching 80% confluence, the cells were washed by 1 mL of PBS thrice, and then incubated with 400 μL of different R123 formulations at the concentration of 5 μM at 37 °C for 4 h. The cells were then washed by ice-cold PBS, followed by the flow cytometry analysis (Guava 6HT, Merck-Millipore). To explore the endocytosis pathways of R123/CG-MEs in MCF-7/ MDR cells, the cells were preincubated with a variety of endocytosis inhibitors for 0.5 h at 37 °C, including 154 mg/mL sucrose (clathrinmediated endocytosis inhibitor), 54 μg/mL genistein (caveolaemediated endocytosis inhibitor), 133 μg/mL amiloride (macropinocytosis inhibitor), 535 μg/mL ammonium chloride (endolyso-
some formation inhibitor), and 0.1% (w/w) sodium azide solution (energy metabolism inhibitor).27,28 Subsequently, the cells were incubated with R123/CG-MEs and R123/C-MEs at the concentration of 5 μM in the presence of the corresponding inhibitor for 2 h, respectively. Afterward, the cells were washed with ice-cold PBS thrice, followed by the flow cytometry analysis. The intracellular delivery and cytoplasmic distribution of R123/CGMEs were monitored using the confocal laser scanning microscope (CLSM). MCF-7/MDR cells were seeded at a density of 1 × 105 cells/ well in a confocal dish (Greiner bio-one) at 37 °C until reaching 60% overspread, and then incubated with R123/CG-MEs and R123/CMEs at the concentration of 5 μM, respectively. At the prearranged time intervals, the cells were washed by ice-cold PBS and then stained with 50 nM LysoTracker Red (Life Technologies) for 30 min at 37 °C. Afterward, the cells were washed by ice-cold PBS thrice and observed immediately using CLSM (TCS SP8, Leica). Mechanism of Inhibition on P-gp-Mediated Efflux. Effect of ECG-MEs on P-gp Expression. To explore whether the treatment with ECG-MEs leads to reduction in the P-gp expression of the MDR cancer cells, the P-gp expression level was quantified using the P-gp antibody binding assay kit (eBioscience).12,29 MCF-7 and MCF-7/ MDR cells were seeded at a destiny of 1 × 105 cells/well in six-well plates, respectively. The cells were washed by PBS twice after reaching 80% overspread and then incubated with 1.5 mL of verapamil (100 μM), G-Rh2 (100 μM), EC-MEs (17 μM etoposide), and ECG-MEs (100 μM G-Rh2) at 37 °C for 2 h, respectively. The following procedure was performed according to the manufacturer’s protocol. The stained cells were determined using the flow cytometry. Effect of ECG-MEs on P-gp ATPase Activity. The Pgp-Glo assay system (Promega) was used to evaluate the potential interaction between G-Rh2 and P-gp ATPase activity. The influence of G-Rh2 and ECG-MEs at G-Rh2 concentration of 100 μM on P-gp ATPase activity was evaluated, and verapamil at a concentration of 200 μM was taken as a control. The following procedures were performed according to the manufacturer’s protocol. The luminescence was detected using the Liquid Chip System (Luminex). In Vitro Cytotoxicity. The cytotoxicity of ECG-MEs against the cancer cells was evaluated using the 3-[4,5-dimethlthiazol-2-yl]-2,5diphenyl tetrazolium bromide (MTT) assay. MCF-7 and MCF-7/ MDR cells at a density of 5 × 103 cells/well were seeded in 96-well plates for 24 h. The culture medium was replaced with 200 μL of different etoposide formulations including the free etoposide suspension, EC-MEs, and ECG-MEs. After incubation for 24 or 48 h, the cells in each well were stained with 20 μL of the MTT PBS solution (5 mg/mL) at 37 °C for 4 h. The medium was then removed, and the resulting formazan crystals were dissolved in 150 μL of dimethyl sulfoxide. The absorbance was measured at 570 nm using the microplate reader (Varioskan Flash, Thermo). Apoptosis-Inducing Capacity. The effect of ECG-MEs on inducing apoptosis of cancer cells was investigated using the Annexin V-PE/7-AAD apoptosis detection kit (Merck-Millipore). Briefly, the cells were seeded at a density of 1 × 105 cells/well in 24-well plates and incubated with the free etoposide suspension, EC-MEs, and ECGMEs for 5 h after reaching 80% overspread, respectively. The following procedures were performed in accordance with the manufacturer’s protocol. The cells were analyzed immediately using the flow cytometry. Animals and Xenograft Tumor Models. All animals were treated in accordance with the Guide for Care and Use of Laboratory Animals, approved by the Animal Experimentation Ethics Committee of Jiangsu Provincial Academy of Traditional Chinese Medicine. Sprague−Dawley (SD) rats (male, 200 ± 20 g), Institute of Cancer Research (ICR) mice (female, 20 ± 2 g), and nude mice (BALB/c, female, 20 ± 2 g) were provided from the Comparative Medicine Center of Yangzhou University. To establish the xenograft tumor models, the mice were subcutaneously inoculated in the back with the MCF-7/MDR cells suspended in 0.1 mL of PBS at a density of 1× 107 cells/mouse. The tumor size was measured by a fine caliper, and the tumor volume (V) C
DOI: 10.1021/acs.biomac.7b00011 Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 2. (A) Particle size and zeta potential of different microemulsion formulations. (B) Hydrodynamic size of ECG-MEs measured by DLS. Inset: TEM image of ECG-MEs; accelerating voltage: 80 kV; scale bar: 50 nm. (C) Encapsulation efficiency of etoposide and G-Rh2 in different microemulsion formulations. (D) Accumulative release rate of etoposide and G-Rh2 from ECG-MEs at pH 7.4 over time. **P < 0.01, compared with etoposide. was calculated as V = L × W2/2, where L and W are the length and width of the tumor, respectively. Oral Absorption and Tumor Targeting. Intestinal Absorption. The oral absorption of ECG-MEs was assessed using in situ intestinal single pass perfusion technique.30 Briefly, different intestinal segments of anesthetized rats, including duodenum, jejunum, ileum, and colon, were exposed under an infrared lamp and catheterized with polypropylene perfusion tubings. The testing perfusates, including the free etoposide suspension, EC-MEs and ECG-MEs at the etoposide concentration of 50 μg/mL, were pumped into four intestinal segments at a flow of 0.22 mL/min, respectively. The perfusates were first used to rinse the intestinal segments for 0.5 h to reach a steady state. The effluent liquid was then collected during additional time periods of 0−0.5 h, 0.5−1 h, 1−1.5 h, and 1.5−2 h, respectively. Afterward, the rats were euthanized, and four intestinal segments were harvested and weighed. The length of the intestinal segments was measured without artificial bending or stretching. The quantity of etoposide in the perfusate was determined using HPLC. The effective permeability coefficient (Peff) was calculated as Peff = −Fin × ln[Cout/Cin × Vout/Vin]/2πrl,30 where Fin represents the flow rate of the perfusate; Cin is the initial concentration of the perfusate; Cout is the concentration of the outlet perfusate after passing the intestinal segment; Vin and Vout are the volumes of the inlet and outlet perfusate, respectively; l is the length of the intestinal segment; and r is the crosssectional radius of the intestinal segment. Intestinal Microdistribution. To monitor the microdistribution of ECG-MEs in the intestinal tract during the intestinal absorption, DiO and DiI, a donor and acceptor fluorescence resonance energy transfer (FRET) pair, were coencapsulated in CG-MEs to obtain DiO/DiI/ CG-MEs, followed by determination of the change in the FRET signals in the intestinal segments. Briefly, the mice were intragastrically administrated with DiO/DiI/CG-MEs at DiO and DiI doses of 50 nmol/kg. At 4 h post-administration, the mice were euthanized. The jejunum segments were collected, followed by cryotomy immediately. The frozen section of jejunum was observed using CLSM. The excitation of the FRET channel was set at 480 nm, and the emission signal was recorded from 580 to 650 nm. The fluorescence intensity was determined using the ImageJ software. The FRET ratio was calculated as [IFRET/(IFRET + IDiO)], where IDiO and IFERT are fluorescence intensities of the DiO signal at 501 nm and the FERT signal at 565 nm upon the DiO excitation wavelength of 480 nm, respectively.31 DiO/CG-MEs without DiI, DiI/CG-MEs without DiO,
and the physical mixture of the free DiO and DiI (DiO+DiI) were taken as references. In Vivo Stability Evaluation. The in vivo stability of ECG-MEs was investigated to estimate whether orally administrated ECG-MEs can maintain the integrity after the intestinal absorption and therefore efficiently accumulate at the tumor site by the nanoparticle-associated EPR effect. DiD and DiR, a donor and acceptor FRET pair, were coencapsulated in CG-MEs to obtain DiD/DiR/CG-MEs, followed by monitoring the change in the FRET signals after oral absorption. Briefly, the mice were intragastrically administrated with DiD/DiR/ CG-MEs at DiD and/or DiR doses of 50 nmol/kg. Images of the isoflurane-anaesthetized mice were taken at 2 and 4 h post administration using the in vivo imaging (IVIS Lumina II). The excitation of the FRET channel was set at 640 nm, and the emission signal was recorded from 720 to 820 nm. Region-of-interests (ROI) were circled around the mice, and the fluorescence intensities were measured using the Living Image Software. The FRET ratio was calculated as [IFRET/(IFRET + IDiD)], where IDiD and IFRET are fluorescence intensities of the DiD signal at 640 nm and the FRET signal at 780 nm upon the DiD excitation wavelength of 640 nm, respectively. DiD/CG-MEs without DiR, DiR/CG-MEs without DiD, and the physical mixture of the free DiD and DiR (DiD+DiR) were taken as references. The DiD signal in the collected plasma after intragastric administration within 24 h was determined using the microplate reader (Varioskan Flash, Thermo). To further evaluate the integrity of ECG-MEs in the blood after oral absorption, the rats were intragastrically administrated with DiD/DiR/ CG-MEs at DiD and DiR doses of 50 nmol/kg, followed by detecting the change in the FRET signals. At prearranged time intervals, the blood samples were collected. The fluorescence intensities of DiD and DiR in the blood were determined. The FRET ratio in the blood was calculated as mentioned above. Biodistribution and Tumor Targeting. The biodistribution and tumor targeting effect of orally administrated microemulsions were evaluated using the in vivo imaging. Cy5-loaded CG-MEs (Cy5/CGMEs) were prepared to track the microemulsions. When the tumor reached around 60 mm3, the mice were intragastrically administrated with Cy5/CG-MEs at Cy5 dose of 30 nmol/kg. Images of the isoflurane-anaesthetized mice were taken at 1, 2, 6, and 12 h postadministration using the IVIS Lumina imaging system. At 24 h after administration, the mice were euthanized. The major normal organs and tumor tissues were harvested and observed. ROI were circled D
DOI: 10.1021/acs.biomac.7b00011 Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 3. (A, B) Cellular uptake of different R123 formulations by Caco-2 cells (A) and MCF-7/MDR cells (B) for 4 h determined using flow cytomerty. **P < 0.01. (C) P-gp expression in untreated MCF-7 cells and MCF-7/MDR cells after treatment with different formulations. **P < 0.01. (D) Stimulation of P-gp ATPase activity in MCF-7/MDR cells after treatment with verapamil, G-Rh2, and ECG-MEs. **P < 0.01, compared with untreated group. around the tissues, and the fluorescence intensities were measured using the Living Image Software. Other Cy5 formulations, including the free Cy5 solution, Cy5/C-MEs without the G-Rh2 component, the physical mixture of Cy5, coix seed oil, and G-Rh2 (Cy5CG mixture), were taken as references. In Vivo Antitumor Efficacy. The in vivo antitumor efficacy of ECG-MEs was evaluated on the MDR tumor models. When the tumor reached around 80 mm3, the MCF-7/MDR-bearing mice were intragastrically administrated with the free etoposide suspension, ECMEs and ECG-MEs at etoposide dosage of 12 mg/kg once daily for 14 days.32 During the treatment, the tumor size and body weight were recorded every day. At day 17, 3 days after the last administration, the tumors were collected, weighed and conserved in 10% neutral buffered formalin. For hematoxylin and eosin (HE) staining, formalin-fixed tumors were embedded in paraffin blocks and visualized using the microscope (VHY-700, Olympus). The apoptosis of tumor cells was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (KeyGen Biotech) according to the manufacturer’s protocol, followed by the fluorescence microscopic observation.28,33 Safety Evaluation. After treatment with either ECG-MEs or saline as a control, the blood samples of the mice were collected. The levels of aspartate transaminase (AST), alanine transaminase (ALT), and blood urea nitrogen (BUN) in the serum samples were assayed using the corresponding assay kits (Amplite) for evaluation on the liver and kidney toxicities.31 The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the serum samples were determined using the enzyme-linked immunosorbent assay (ELISA) kit (KeyGen Biotech). The liver and spleen were collected and weighed, and the liver and spleen indices were calculated as W/W0, where W and W0 are the liver or spleen weight and body weight after treatment, respectively.34 The
histological investigations of the normal organs were conducted using the HE staining. Data Analysis. Data are given as the mean ± standard deviation. Statistical significance was tested by two-tailed Student’s t test, and considered as *P < 0.05 and **P < 0.01, respectively.
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RESULTS AND DISCUSSION
Preparation and Characterization. A “one-step emulsion” method was applied to prepare ECG-MEs, which has many advantages of simple operation and readily functional modification.25 The obtained ECG-MEs containing etoposide, coix seed oil, and G-Rh2 (1:80:6, w/w/w) had an average particle size of 46.3 nm, PDI of 0.114 and a zeta potential of −15.8 mV (Figure 2A), which had no significant difference with EC-MEs without the G-Rh2 component, CG-MEs without loading etoposide, and the blank coix seed oil microemulsions (C-MEs). The data suggest that the introduction of etoposide and G-Rh2 did not influence the particle size and surface charge of the microemulsion at such determined ratio. The morphology of ECG-MEs characterized by TEM displayed a spherical shape with a narrow size dispersity (Figure 2B), which was consistent with the hydrodynamic size measured by DLS. EE of etoposide and G-Rh2 were both higher than 90% (Figure 2C), indicating that the functional agents were almost entirely encapsulated inside the coix seed oil-based microemulsions. In addition, due to the excipient-like property of coix seed oil and G-Rh2, the conventional oil phase and surfactant in ECG-MEs was reduced by 100% and 7.5% in comparison with etoposideloaded microemulsion (E-MEs), respectively (Table S1). E
DOI: 10.1021/acs.biomac.7b00011 Biomacromolecules XXXX, XXX, XXX−XXX
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etoposide and coix seed oil was sufficient for interfering the function of P-gp and thereby overcoming the P-gp-mediated efflux of anticancer components. For MCF-7 cells, although different microemulsion formulations significantly improved the endocytosis of R123, neither G-Rh2 nor verapamil was able to remarkably influence the intracellular accumulation of R123 (Figure S3A), further indicating the significances of G-Rh2functionalized microemulsion in the MDR reversal. To further exploit the potential mechanism of P-gp inhibition effect of G-Rh2 and the obtained G-Rh2-containing microemulsion, the change in the P-gp expression and the activity of the P-gp ATPase were detected after treatment. The P-gp expression level of MCF-7/MDR cells after different treatments was first tested (Figure 3C). As expected, higher P-gp expression was found on MCF-7/MDR cells compared with MCF-7 cells. No significant variation in the P-gp expression level of MCF-7/MDR cells was found after the treatment of either G-Rh2 or ECG-MEs. The data indicate that the antiresistance effect of ECG-MEs as P-gp inhibition does not result from reducing the P-gp expression. Next, we evaluated the effect of ECG-MEs on the activity of P-gp ATPase (Figure 3D). Verapamil, the P-gp inhibitor, can stimulate the activity of the P-gp ATPase. Compared with the untreated group, the positive ΔRLU (relative light unit) value after treatment with verapamil suggests a strong activating effect on the ATPase of P-gp. The ΔRLU value after treatment with G-Rh2 was comparable to that after treatment with verapamil, exhibiting a potent capability of consuming ATP of P-gp by G-Rh2 similar to that of verapamil. Moreover, ECG-MEs containing G-Rh2 showed the effect on depleting ATP by G-Rh2-activating P-gp. These results suggest that the potential mechanism of P-gp inhibition by ECG-MEs was associated with the stimulation of the P-gp ATPase activity. The enhanced intracellular accumulation of etoposideinvolving P-gp substrates delivered by ECG-MEs was not only attributed to the P-gp inhibition of the G-Rh2 component, but also due to the P-gp bypassing action by endocytosis of intact ECG-MEs.12,29,37 We determined the potential internalization pathway of ECG-MEs using specific endocytosis inhibition method. The change in the cellular uptake of R123/CG-MEs was detected in the presence of different endocytotic inhibitors (Figure S3B). The presence of sodium azide caused the decreased cellular uptake of R123/CG-MEs and R123/C-MEs, indicating both of them were internalized into cells in an active transportation manner related to energy expenditure. After treatment with ammonium chloride, a cell lysosomotropic agent, the intracellular amount of R123/CGMEs significantly reduced compared with that of untreated, but higher than that of R123/C-MEs, which suggest that the introduction of the G-Rh2 component in the R123/CG-MEs formulation could partially alleviate the endolysosomal entrapment.38,39 Moreover, the presence of sucrose and genistein notably decreased the cellular uptake of R123/CG-MEs, demonstrating that R123CG-MEs might enter the cells through both caveolae-mediated and clathrin-mediated endocytosis pathway. Furthermore, the intracellular distribution of R123/ CG-MEs was observed using CLSM. The yellow fluorescence represented the colocalization between the green fluorescence of the R123-loaded microemulsion and the red fluorescence of the LysoTracker Red-labeled lysosomes, which is indicative of the endolysosomal entrapment of the microemulsions. When the cells were incubated with R123/C-MEs without the G-Rh2 component for 4 h, the overwhelming yellow fluorescence was
To validate our assumption that G-Rh2 would be released prior to etoposide from ECG-MEs, the release profiles of etoposide and G-Rh2 were determined at pH 7.4 (Figure 2D). The cumulative release amount of G-Rh2 was about 18% within the first 4 h, which was significantly faster than that of etoposide with only 6% released. After 24 h, 41% of G-Rh2 was released from ECG-MEs, which was 2.3-fold that of etoposide. By comparison, no significant difference was observed in the release of etoposide and G-Rh2 from the physical ECG mixture (Figure S1A). These results suggest that spatial control of etoposide and G-Rh2 in different layers of microemulsions could realize a desired release property, leading to the initialstaged P-gp inhibition by G-Rh2 and subsequent synergistic effect of etoposide, coix seed oil, and G-Rh2. The most potential explanation for such time-staggered release characteristic is that most of etoposide is coexisted with coix seed oil that is localized in the core of microemulsion through the hydrophobic force, while G-Rh2 was prone to distribute in the surfactant layer as the shell of microemulsion due to the inherent amphipathy and good compatibility with RH40. Moreover, the release profiles of ECG-MEs in the simulated gastric and intestinal fluids were also investigated using the similar method. The time-staggered release tendency of ECGMEs was also found (Figure S1B,C), which was similar to that at pH 7.4. The cumulative release amounts of etoposide and GRh2 released from ECG-MEs in the simulated gastric fluid were less than 10% and 20% within 2 h, respectively, indicating that ECG-MEs had a high stability in the gastrointestinal tract. In addition, the particle size and zeta potential of ECG-MEs had no significant change under dilution of PBS with the pH values ranging from 4.5 to 8.0, further confirming a high stability of ECG-MEs (Figure S2). Cellular Uptake and Intracellular Delivery. To validate the effect of G-Rh2 as a P-gp inhibitor on enhancing the cellular uptake of intestinal epithelial cells and MDR tumor cells, R123, a P-gp substrate was loaded in the microemulsion, and the intracellular accumulation of R123/CG-MEs in different cells was determined using the flow cytometry. As shown in Figure 3A, the presence of verapamil, a typical P-gp inhibitor, increased the amount of R123 in Caco-2 cells that are commonly used to resemble the enterocytes with P-gp expression.12,35 Similarly, the cellular uptake of R123 increased in the presence of G-Rh2, implying the ability of G-Rh2 to inhibit the P-gp efflux. R123/CG-MEs showed the strongest effect on elevating the cellular uptake of R123, but no further enhancement on intracellular accumulation was found when additional G-Rh2 was added into R123/CG-MEs. These results indicate that introduction of G-Rh2 into the formulation of microemulsion was favorable to increase uptake of intestinal cells and thereby strengthen the oral absorption of anticancer drugs. For the evaluation on overcoming the MDR of tumor cells by ECG-MEs, the cellular uptake of R123/CG-MEs were evaluated on MCF-7/MDR cells. The presence of either G-Rh2 or verapamil significantly increased the R123 accumulation in MCF-7/MDR cells due to the respective P-gp inhibition capabilities. The cellular uptake of R123/C-MEs was 2.4-fold higher than that of free R123, which could be attributed to the inherent enhancement of nanosized drug delivery system on cellular uptake.36 Note that, the cellular uptake of R123/CGMEs presented 4.6- and 1.9-fold that of R123 and R123/CMEs, respectively (Figure 3B), which suggest that the initialstaged release of G-Rh2 from the microemulsion prior to F
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Table 1. IC50 and Resistance Reversion Index (RRI) of Different Formulations against MCF-7 and MCF-7/MDR Cells for 24 and 48 h MCF-7 cells
a
MCF-7/MDR cells
formulations
24 h, IC50 (μg/mL)
48 h, IC50 (μg/mL)
24 h, IC50 (μg/mL)
etoposide EC-MEs ECG-MEs
27.1 ± 3.1 16.1 ± 2.2 9.3 ± 0.6
13.5 ± 1.6 7.3 ± 0.4 7.3 ± 0.5
38.9 ± 4.4 29.0 ± 3.4 3.3 ± 0.4
RRIa
48 h, IC50 (μg/mL)
RRI
1.3 11.8
13.8 ± 1.2 12.9 ± 0.9 1.3 ± 0.2
1.1 10.6
RRI = IC50 (etoposide)/IC50 (test formulations).
Figure 4. (A, B) Antiproliferative activity of different etoposide formulations on MCF-7/MDR cells for 24 h (A) and 48 h (B). **P < 0.01, *P < 0.05. (C) Apoptosis ratio of MCF-7/MDR cells after treatment with different etoposide formulations. In each panel, the lower-right and upper-right quadrants represent the populations of early apoptotic cells and late apoptotic cells, respectively. The average population (%) in each quadrant is indicated by the numbers.
observed, demonstrating that most of R123C-MEs were entrapped in the late endosomes and lysosomes after internalization. In sharp contrast, the yellow part was significantly weaker in the R123/CG-MEs group, as judged by the obvious separation between the green and red fluorescence at different incubation periods of time (Figure S4), indicating that G-Rh2 could potentially suppress the endolysosomal trapping and facilitate the intracellular delivery of anticancer drugs. Accordingly, the results suggest that integrating G-Rh2 into ECG-MEs is favorable to realize multicomponent release in the cytoplasm and exert optimal therapeutic effects against the MDR cancer cells through interfering the P-gp ATPase activity and reducing the endolysosomal entrapment. In Vitro Cytotoxicity. The in vitro cytotoxicity of different etoposide formulations against MCF-7 and MCF-7/MDR cells were evaluated using the MTT assay. Both EC-MEs and ECG-
MEs showed higher cytotoxicity against MCF-7 cells than the free etoposide suspension (Figure S5A). The half inhibitory concentration (IC50) of EC-MEs and ECG-MEs were about 16.1 and 9.3 μg/mL for 24 h of treatment, showing higher cytotoxicity than the free etoposide (Table 1). However, the introduction of G-Rh2 did not show obvious improvement of cytotoxicity on MCF-7 cells model especially for 48 h of treatment (Figure S5B). There is no significant enhancement on the antiproliferation against MCF-7 cells for 48 h between ECG-MEs and EC-MEs with comparable IC50 values about 7.3 μg/mL (Table 1). By comparison, ECG-MEs exhibited the strongest cytotoxicity toward MCF-7/MDR cells for 24 h (Figure 4A), which had the IC50 value of 3.3 μg/mL, 10.8-fold and 7.8-fold lower than that of etoposide and EC-MEs, respectively. The data suggest that the presence of G-Rh2 in the microemulsion contributes to inhibiting the P-gp-mediated efflux of etoposide by initial-staged release from ECG-MEs, G
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Table 2. Permeate Coefficient (Peff) of Different Intestinal Segments after Perfusion with Different Etoposide Formulations Containing 50 μg/mL Etoposide
a
formulations
duodenum (×10−4 cm/s)
jejunum (×10−4 cm/s)
ileum (×10−4 cm/s)
colon (×10−4 cm/s)
etoposide EC-MEs ECG-MEs
2.09 ± 0.19 3.59 ± 0.61a 4.05 ± 0.37a
2.24 ± 0.63 3.68 ± 0.56a 4.26 ± 0.43a
1.84 ± 0.36 3.27 ± 0.69a 3.66 ± 0.72a
0.70 ± 0.45 2.15 ± 0.31a 2.21 ± 0.28a
P < 0.01, compared with etoposide.
Figure 5. (A) CLSM images of jejunum sections of the mice after intragastric administration with different formulations for 4 h. Scale bar: 75 μm. (B) FRET ratios qualified by the fluorescence intensity in the whole jejunum section. **P < 0.01. (C) FRET ratio in the blood of the mice after intragastric administration with DiO/DiI/CG-MEs and the physical mixture of DiO and DiI over time.
resulting in a stronger antiproliferative effect. Moreover, the resistance reversion index (RRI) of ECG-MEs was calculated to be 11.8 after treating MCF-7/MDR cells for 24 h, providing a concrete proof of feasibility of anti-MDR (Table 1). When ECG-MEs were incubated with MCF-7/MDR cells for an additional 24 h, the IC50 value logically decreased to 1.3 μg/mL
(Figure 4B). The combined index of ECG-MEs against MCF7/MDR cells for 24 and 48 h was calculated to be 0.92 and 0.59, respectively. These results further indicate that the ECGMEs formulation could act as a tool to integrate respective advantages of various anticancer agents, leading to a more effective P-gp inhibition and a stronger cytotoxicity through H
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Figure 6. (A, B) Fluorescence images of the mice after intragastric administration with different formulations for 2 h (A) and 4 h (B). (C) FRET ratios qualified by the fluorescence intensity in the whole mouse body. **P < 0.01, compared with the physical mixture of DiD and DiR. (D) The change in the DiD signal in the plasma of the mice after intragastric administration with DiO/DiI/CG-MEs over time.
Oral Absorption and In Vivo Stability. The oral absorption of ECG-MEs was assessed using in situ intestinal single pass perfusion technique. As shown in Table 2, The Peff of ECG-MEs was calculated to be 4.05 × 10−4 and 4.26 × 10−4 cm/s in the duodenum and jejunum segments, which were 1.94- and 1.90-fold that of the free etoposide suspension, respectively. The data suggest that ECG-MEs greatly improved the intestinal absorption due probably to the prolonged retention in the intestinal tract. This absorption-enhancing capability of ECG-MEs was still observed in the ileum and colon segments, although the absorption reduced obviously in both intestinal segments. In addition, EC-MEs displayed the similar enhancement on intestinal absorption compared with etoposide, but moderately lower than ECG-MEs, indicating a G-Rh2-mediated P-gp inhibition. The integrity of ECG-MEs after crossing intestinal barrier is of great importance to the following blood circulation and tumor accumulation by the EPR effect. We applied the FRET
sequential release of G-Rh2, as well as the enhancement on cellular uptake and intracellular transport. Apoptosis-Inducing Capacity. We investigated whether the incorporation of G-Rh2 could further enhance the apoptosis-inducing capacity of the microemulsions on the MDR cancer cells. As shown in Figure 4C, both etoposide and EC-MEs showed apoptosis-inducing effect on MCF-7/MDR cells. The total apoptosis ratio (early apoptosis ratio plus late apoptosis ratio) of MCF-7/MDR cells were 29.9 and 40.8% after 5 h of treatment with etoposide and EC-MEs, respectively. By comparison, ECG-MEs displayed 1.26- and 2.09-fold increase in induction of cell apoptosis compared with ECMEs and etoposide, respectively, which showed an obvious merit of combination therapy. However, the enhancement of ECG-MEs on apoptosis induction on MCF-7 cells was not greater than that on MCF-7/MDR cells, which implied that GRh2 possesses no remarkable contribution on enhancing cellular uptake of ECG-MEs on MCF-7 cells (Figure S5C). I
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Figure 7. (A) Fluorescence images of the MCF-7/MDR tumor-bearing mice after intragastric administration with different Cy5 formulations. The circles indicate the tumor sites. (B) Fluorescence images of tumors and different normal tissues of MCF-7/MDR tumor-bearing nude mice at 24 h post-intragastric administration. (C) Quantification of the fluorescent signals from the tumors and different normal tissues using ROI analysis. **P < 0.01.
technique to evaluate the structure stability of microemulsion during adhesion with the intestinal wall. That the FRET effect weakens or disappears indicates the integrity of microemulsion is destroyed. The jejunum segments were collected after administration with different formulations for 4 h (Figure 5A). Importantly, the fluorescence signal of DiO/DiI/CG-MEs in the FRET channel was obviously determined, suggesting that DiO/DiI/CG-MEs might cross the intestinal tract and enter into blood as intact nanoparticles. In contrast, in the physical mixture of DiO and DiI, the fluorescence signals of DiO and DiI were only observed at the respective excitation wavelengths, and the FRET effect was hardly found. The FRET ratio of DiO/DiI/CG-MEs was approximately 0.4 (Figure 5B), notably higher than other formulations. We further estimated the stability of DiO/DiI/CG-MEs after intragastric administration by monitoring the change in the FRET ratio in the blood (Figure 5C). The FRET ratio of DiO/DiI/CG-MEs in the blood samples remained stable within 24 h after intragastric administration. These data demonstrate that a significant part
of microemulsion showed highly stable during the process of intestinal absorption and blood circulation. Furthermore, we coencapsulated another FRET pair, DiD and DiR into the microemulsion to obtain DiD/DiR/CG-MEs, and detected the change in the FRET signal of DiD/DiR/CGMEs after intragastric administration using the in vivo imaging technique (Figure 6A). The monoloading formulation, either DiD/CG-MEs or DiR/CG-MEs, could only be visualized at the respective channels at 2 h post-injection. In sharp contrast, the FRET signal of DiD/DiR/CG-MEs was apparently observed, in addition to the obvious respective fluorescence signal. As expected, the physical mixture of DiD and DiR, DiD + DiR had no significant FRET effect due to the long distance between two dyes in the whole body. The FRET phenomenon of DiD/ DiR/CG-MEs maintained as the time extended to 4 h (Figure 6B), which was similar to the trend at 2 h. The quantitative results showed that the FRET ratios of DiD/DiR/CG-MEs at 2 and 4 h were both higher than 0.5 (Figure 6C), which were about 8-fold that of the physical mixture of DiD and DiR. J
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Figure 8. (A) Tumor growth curve of the MCF-7/MDR tumor-bearing mice after intragastric administration with different etoposide formulations at etoposide dose of 12 mg/kg for 14 days (once daily). The arrow represents the time of intragastrical administration. **P < 0.01. (B) The tumor index of MCF-7/MDR tumor-bearing mice after treatment at 17 days post-administration. The tumor index is represented by the ratio of the tumor weight and the body weight. **P < 0.01. (C) Kaplan−Meier survival curves of the MCF-7/MDR tumor-bearing mice after treatment. (D) Change in the body weight of MCF-7/MDR tumor-bearing mice after treatment. (E) Images of the HE-stained tumor sections after treatment. Scale bar: 100 μm. (F) Fluorescence images of the tumor sections stained with FITC-dUTP after treatment. The nuclei were stained with DAPI. Scale bar: 100 μm.
Additionally, the pharmacokinetics of DiD/DiR/CG-MEs after intragastric administration was also evaluated by detecting the DiD signal in the plasma within 24 h (Figure 6D). The twopeak DiD signal of DiD/DiR/CG-MEs was determined at 0.5 and 4 h post-administration, respectively, which might be attributed to the involvement of hepato-enteric circulation. These results further indicate a stable structure of microemulsion in the whole body for a potential enhancement on oral bioavailability. Biodistrubution and Tumor Targeting. The biodistribution of different Cy5-labeled formulations was investigated using the in vivo imaging after intragastrical administration into the MCF-7/MDR tumor-bearing mice (Figure 7A). At 1 h post-administration, all the formulations exhibited strong fluorescence signals in the whole body. When the time increased to 2 h, Cy5/CG-MEs showed an obviously stronger fluorescence signal at the tumor site compared with Cy5/C-
MEs, indicating the G-Rh2-mediated promotion on the intestinal absorption and the accumulation of anticancer drug at the tumor site. Although the fluorescence signals reduced in all test groups at 6 h post-administration, Cy5/CG-MEs still displayed the greatest fluorescence distribution in the tumor region. And such tumor accumulation was even found at 12 h. Furthermore, the ex vivo imaging was observed to validate the specific accumulation at the end of the observation time. The fluorescence intensity of Cy5/CG-MEs at the tumor site was notably higher than that of other formulations (Figure 7B). For the quantitative analysis, the greatest fluorescence intensity of Cy5 was determined in the tumor compared with other formulations (Figure 7C). In addition, the highest fluorescence intensity of Cy5/CG-MEs in the intestine at 24 h postadministration further confirmed that Cy5/CG-MEs had an enhanced retention capability in the intestines for improving the oral absorption of the cargos. The results suggest that ECGK
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MEs might be safe as an oral anticancer drug delivery system in vivo.
MEs could efficiently cross the intestinal barrier via the intact nanoparticle and accumulate at the tumor site by the EPR effect. In Vivo Antitumor Efficacy. The in vivo antitumor efficacy of ECG-MEs was evaluated using the MCF-7/MDR tumor xenograft mice. After intragastric administration once every day at the etoposide dose of 12 mg/kg for 14 days, the tumor growth was obviously suppressed by different etoposide formulations compared with saline (Figure 8A). ECG-MEs showed the greatest effect on inhibiting the tumor growth compared with other etoposide formulations. ECG-MEs had a tumor growth inhibition rate40 of 70.7% compared with 36.1% of EC-MEs (Figure 8B), indicating that sequential drugs release played an important role in the first-staged P-gp inhibition and consequent more therapeutics accumulation in the MDR cancer cells. Treatment of ECG-MEs significantly prolonged the survival of MCF-7/MDR tumor-bearing mice compared with other etoposide formulations (Figure 8C). In addition, the body weight did not change obviously during the treatment with ECG-MEs (Figure 8D). Furthermore, the histologic alternation of the tumor after applying various etoposide formulations was investigated using the HE staining method (Figure 8E). Obvious cancer cells remission was found in the tumor tissue at 72 h after the last treatment (Day 24) with ECG-MEs, further confirming its most prominent therapeutic activity in vivo. Moreover, the results obtained using the in situ TUNEL assay showed the highest level of cell apoptosis in the tumor tissues of the mice treated with ECG-MEs (Figure 8F), suggesting that the strongest tumor growth suppression was attributable in part to the increased apoptosis-inducing effect of ECG-MEs. Taken together, these results indicate that orally administrated ECGMEs are capable of prolonging the retention in the intestine, promoting the oral absorption as the intact nanoparticle form, transporting to the tumor by the nanoparticle-related EPR effect, and increasing the intracellular accumulation of etoposide by the initial-staged release of G-Rh2 with P-gp inhibition in the MDR cancer cells for overcoming the MDR and enhancing the antitumor efficacy. Safety Evaluation. A certain of serious side effects after oral administration of etoposide, including myelosuppression, liver and kidney damage and alimentary canal toxicity, have been commonly reported in various clinical researches.41−43 Our previous study has pointed out that combination of coix seed oil with chemotherapeutic agents was able to alleviate the above-mentioned problems.44 In view of this, the safety evaluation after treatment with ECG-MEs was performed by monitoring the variation in the physiological and biological indices, including the liver and spleen indices, the concentrations of cytokines (TNF-α and IL-6), liver enzymes (ALT and AST), and acute renal lesion indicators (BUN), and the histopathology of major organs. No significant change in both liver and spleen indices was determined after treatment with ECG-MEs compared with saline (Figure S6A). The serum concentration of TNF-α and IL-6 at 72 h post-administration of ECG-MEs was comparable to that after administration of saline (Figure S6B). As for the ALT, AST, and BUN concentrations in the serum, all the test groups were within the normal ranges (Table S2). Additionally, the histopathological analysis using the HE staining presented that no remarkable difference in the in normal organs (Figure S6C). No obvious solid lesion or abnormality was observed in the HE-stained sections of heart, liver, spleen, lung, and kidney. These data suggest that ECG-
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CONCLUSIONS We developed multicomponent microemulsions, ECG-MEs, for oral delivery of etoposide to treat MDR breast cancer, which consisted of etoposide, G-Rh2, and coix seed oil. Orally administrated ECG-MEs could cross intestinal barrier and circulate in the blood in a manner of intact nanoparticles, leading to enhanced blood persistence and tumor accumulation. The spatiotemporal controlled release property of ECGMEs resulted in the efficient P-gp inhibition by the initialreleased G-Rh2 and the increased intracellular accumulation by the sequential-released etoposide. Synergistic antitumor activity of etoposide, G-Rh2, and coix seed oil using ECG-MEs was validated on the xenograft MDR tumor mouse models. The results suggest that ECG-MEs could be applied as a safe and efficient oral delivery system of anticancer drug for the MDR breast cancer therapy.
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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.biomac.7b00011. Composition of formulations, additional characterization of drug release, stability of microemulsions in different simulated physiological environments, mechanism of cellular uptake, intracellular localization, cytotoxicity, and safety evaluation in vivo (PDF).
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Ran Mo: 0000-0003-4010-8879 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported financially by the National Natural Science Foundation of China (81503264) and the Natural Science Foundation of Jiangsu Province of China (BK20141038, BK20151048). The work was also supported by the Natural Science Foundation of Jiangsu Province of China for Distinguished Young Scholars (BK20150029), the Young Talents Promoting Program of CAST, the Six Talent Peaks Project of Jiangsu Province of China, and the Jiangsu Specially-Appointed Professors Program to R.M.
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ABBREVIATIONS MDR, multidrug resistance; P-gp, P-glycoprotein; EPR, enhanced permeability and retention; G-Rh2, ginsenoside Rh2; ECG-MEs, etoposide and ginsenoside Rh2 coloaded coix seed oil microemulsions; EC-MEs, etoposide-loaded coix seed oil microemulsions; C-MEs, coix seed oil microemulsions; E-MEs, etoposide-loaded microemulsions; DLS, dynamic light scattering; TEM, transmission electron microscope; PBS, phosphate buffer saline; EE, encapsulation efficiency; PB, phosphate buffer; CG-MEs, G-Rh2-containing coix seed oil L
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(25) Qu, D.; Ma, Y.; Sun, W.; Chen, Y.; Zhou, J.; Liu, C.; Huang, M. Int. J. Nanomed. 2015, 10, 1173−1187. (26) Assanhou, A. G.; Li, W.; Zhang, L.; Xue, L.; Kong, L.; Sun, H.; Mo, R.; Zhang, C. Biomaterials 2015, 73, 284−295. (27) Su, Z.; Xing, L.; Chen, Y.; Xu, Y.; Yang, F.; Zhang, C.; Ping, Q.; Xiao, Y. Mol. Pharmaceutics 2014, 11, 1823−1834. (28) Mo, R.; Jiang, T.; DiSanto, R.; Tai, W.; Gu, Z. Nat. Commun. 2014, 5, 3364. (29) Jin, X.; Mo, R.; Ding, Y.; Zheng, W.; Zhang, C. Mol. Pharmaceutics 2014, 11, 145−157. (30) Valicherla, G. R.; Dave, K. M.; Syed, A. A.; Riyazuddin, M.; Gupta, A. P.; Singh, A.; Wahajuddin; Mitra, K.; Datta, D.; Gayen, J. R. Sci. Rep. 2016, 6, 26895. (31) Sun, Q.; Kang, Z.; Xue, L.; Shang, Y.; Su, Z.; Sun, H.; Ping, Q.; Mo, R.; Zhang, C. J. Am. Chem. Soc. 2015, 137, 6000−6010. (32) Shete, H.; Sable, S.; Tidke, P.; Selkar, N.; Pawar, Y.; Chakraborty, A.; De, A.; Vanage, G.; Patravale, V. Biomaterials 2015, 57, 116−132. (33) Jiang, T.; Sun, W.; Zhu, Q.; Burns, N. A.; Khan, S. A.; Mo, R.; Gu, Z. Adv. Mater. 2015, 27, 1021−1028. (34) Mo, R.; Sun, Q.; Xue, J.; Li, N.; Li, W.; Zhang, C.; Ping, Q. Adv. Mater. 2012, 24, 3659−3665. (35) Mo, R.; Xiao, Y.; Sun, M.; Zhang, C.; Ping, Q. Int. J. Pharm. 2011, 409, 38−45. (36) Hong, W.; Chen, D.; Zhang, X.; Zeng, J.; Hu, H.; Zhao, X.; Xiao, M. Biomaterials 2013, 34, 9602−9614. (37) Qin, J. J.; Wang, W.; Sarkar, S.; Zhang, R. J. Controlled Release 2016, 237, 101−114. (38) Zhang, B.; Mallapragada, S. Acta Biomater. 2011, 7, 1580−1587. (39) Panyam, J.; Zhou, W. Z.; Prabha, S.; Sahoo, S. K.; Labhasetwar, V. FASEB J. 2002, 16, 1217−1226. (40) Qu, D.; Sun, W.; Liu, M.; Liu, Y.; Zhou, J.; Chen, Y. Int. J. Pharm. 2016, 503, 90−101. (41) Yamaguchi, M.; Kwong, Y. L.; Kim, W. S.; Maeda, Y.; Hashimoto, C.; Suh, C.; Izutsu, K.; Ishida, F.; Isobe, Y.; Sueoka, E.; Suzumiya, J.; Kodama, T.; Kimura, H.; Hyo, R.; Nakamura, S.; Oshimi, K.; Suzuki, R. J. Clin. Oncol. 2011, 29, 4410−4416. (42) Pein, F.; Tournade, M. F.; Zucker, J. M.; Brunat-Mentigny, M.; Deville, A.; Boutard, P.; Dusol, F.; Gentet, J. C.; Legall, E.; Mechinaud, F. J. Clin. Oncol. 1994, 12, 931−936. (43) Vanhoefer, U.; Rougier, P.; Wilke, H.; Ducreux, M. P.; Lacave, A. J.; Van Cutsem, E.; Planker, M.; Santos, J. G.; Piedbois, P.; Paillot, B.; Bodenstein, H.; Schmoll, H. J.; Bleiberg, H.; Nordlinger, B.; Couvreur, M. L.; Baron, B.; Wils, J. A. J. Clin. Oncol. 2000, 18, 2648− 2657. (44) Qu, D.; He, J.; Liu, C.; Zhou, J.; Chen, Y. Int. J. Nanomed. 2014, 9, 109−119.
microemulsions; R123/C-MEs, Rhodamine 123-loaded coix seed oil microemulsions; CLSM, confocal laser scanning microscope; MTT, 3-[4,5-dimethlthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; DiD/DiR/CG-MEs, DiD and DiR coloaded CG-MEs; DiO/DiI/CG-MEs, DiO and DiI coloaded CG-MEs; ICR, Institute of Cancer Research; FRET, fluorescence resonance energy transfer; HE, hematoxylin and eosin; AST, aspartate transaminase; ALT, alanine transaminase; BUN, blood urea nitrogen; TNF-α, tumor necrosis factor-α; IL6, interleukin-6; TUNEL, transferase dUTP nick end labeling
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DOI: 10.1021/acs.biomac.7b00011 Biomacromolecules XXXX, XXX, XXX−XXX
