Reversal of Multidrug Resistance by Mitochondrial Targeted Self

Article pubs.acs.org/molecularpharmaceutics

Reversal of Multidrug Resistance by Mitochondrial Targeted SelfAssembled Nanocarrier Based on Stearylamine Zhiwen Zhang,† Zeying Liu,‡ Li Ma,† Shijun Jiang,‡ Yixin Wang,† Haijun Yu,*,† Qi Yin,† Jingbin Cui,‡ and Yaping Li*,† †

Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China

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ABSTRACT: Multidrug resistance (MDR) remains one of the major challenges for successful chemotherapy. Herein, we tried to develope a mitochondria targeted teniposide loaded self-assembled nanocarrier based on stearylamine (SA-TSN) to reverse MDR of breast cancer. SA-TSN was nanometer-sized spherical particles (31.59 ± 3.43 nm) with a high encapsulation efficiency (99.25 ± 0.21%). The MDR in MCF-7/ADR cells was obviously reduced by SA-TSN, which mainly attributed to the markedly reduced expression of P-gp, increased percentages in G2 phase, selectively accumulation in mitochondria, decrease of mitochondrial membrane potential, and greatly improved apoptosis. The plasma concentration of teniposide was greatly improved by SA-TSN, and the intravenously administered SA-TSN could accumulate in the tumor site and penetrate into the inner site of tumor in MCF-7/ADR induced xenografts. In particular, the in vivo tumor inhibitory efficacy of SA-TSN in MCF-7/ADR induced models was more effective than that of teniposide loaded self-assembled nanocarrier without stearylamine (TSN) and teniposide solution (TS), which verified the effectiveness of SA-TSN in reversal of MDR. Thereby, SA-TSN has potential to circumvent the MDR for the chemotherapy of breast cancer. KEYWORDS: stearylamine, multidrug resistance, self-assembly, nanocarrier, teniposide, mitochondria

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INTRODUCTION Multidrug resistance (MDR) remains one of the major challenges and causes of mortality in the treatment of cancer.1,2 In clinical trials, MDR causes the failure of cancer treatment for over 90% of patients.3 Typically, the sensitivity of cancer cells to cytotoxic drugs are severely reduced by MDR, and chemotherapeutic effects can be obviously attenuated thereof. Moreover, the MDR of cancer cells never stops evolving even under the pressure of a drug treatment.4 Thereby, it is great necessary to exploit effective strategies to circumvent the MDR of cancer cells in chemotherapy. Generally, MDR includes the intrinsic and acquired resistance through exposure of chemotherapeutic agents to cancers.5−7 The intrinsic resistance is caused by genetic and epigenetic changes of cancer cells through altering function of pro-apoptotic or apoptotic genes encoded proteins and caspase proteins involved in the apoptosis signaling pathway.7−10 The intrinsic MDR is mainly associated with mitochondria, which plays a central role in tumor apoptosis and energy metabolism.7,8,11,12 Moreover, mitochondria are the emerging target of anticancer drugs, and it was reported that a mitochondrial targeting compound of vitamin E succinate could significantly improve its pro-apoptotic and anticancer activity.11−14 Meanwhile, the mitochondria targeted delivery of anticancer drugs with liposomes could enhance the in vitro and in vivo antitumor activity.15−17 Thereby, the selectively targeted delivery of chemotherapeutic drugs to mitochondria could have © XXXX American Chemical Society

potential to reverse the MDR of resistant cancer cells, which has been seldom investigated up to date. On the other hand, the acquired resistance of cancer can mainly result from the overexpression of drug efflux pumps of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein (MRP), modifications in drug metabolism via glutathione-S-transferase or cytochrome P450 activity, alterations in DNA repair mechanisms, and modifications of apoptotic signaling.1,2,6 Therein, the most characterized mechanism of acquired MDR is the increased drug efflux through overexpressed P-gp efflux pumps, which can be effectively inhibited by some functional amphiphilic polymers.18−20 Several polymeric nanoparticles or micelles have been shown to enhance the cellular uptake of anticancer drugs in resistant cancer cells and reverse MDR by inhibiting the overexpression of P-gp.20−22 Therefore, the nanobased drug delivery system with functional materials is promising to reduce the efflux of anticancer drugs and overcome the acquired MDR for cancer therapy. Stearylamine (SA) is a lipophilic cationic molecule with a primary amine group. SA modified cationic liposome could induce the cell apoptosis via the mitochondria mediated Received: January 22, 2013 Revised: March 27, 2013 Accepted: April 30, 2013

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dx.doi.org/10.1021/mp400039j | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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pathway.23,24 In the present work, we try to develope a nanocarrier based on SA and some functional amphiphiles with P-gp inhibition effects for mitochondrial targeted delivery of anticancer drugs to reduce the intrinsic and acquired MDR in chemotherapy, which has not been investigated so far. Teniposide, a typical DNA-binding agent used in the treatment of breast cancer, leukemia, lymphoma, intracranial malignant tumor, and some other types of cancer,25−27 was selected as a model drug, which exhibits severe drug resistance in tumor cells due to the overexpressed transporters proteins of P-gp and MRP, and decreased topoisomerase II expression.28,29 In addition, D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and Solutol HS-15 are usually utilized for the reversal of MDR.30 Herein, a teniposide loaded self-assembled nanocarrier based on SA (SA-TSN) was designed, and the possible reversal mechanism of MDR by SA-TSN was investigated by in vitro and in vivo evaluations.

loaded self-assembled nanocarrier without SA (TSN), which was composed of teniposide, TPGS, MCT, and HS-15 (1:20:15:10, w/w), was prepared according to the same procedure as SA-TSN. In addition, self-assembled nanocarrier without SA and teniposide (SN) and SA contained selfassembled nanocarrier without teniposide (SA-SN) were prepared in the same procedure. The morphology of SA-TSN was determined under a transmission electronic microscopy (TEM, JEOL JEM-1230, Japan). The size distribution was measured by dynamic light scattering on a Nano ZS 90 instrument (Malvern, UK). To clarify the crystalline state of teniposide in SA-TSN, the X-ray diffraction (XRD) patterns were recorded on an XRD-6000 Xray diffractometer (Shimadzu, Tokyo, Japan) in the range of 3− 50° at a step size of 0.02°. The XRD measurements of free teniposide and SA powder also were performed as a control. The encapsulation efficiency (EE) was estimated according to the following formula: EE = (Wt − Wf)/Wt × 100%, therein Wt referred to the total amount of teniposide and Wf was that of free drug. Free teniposide was separated from SA-TSN by ultrafiltration with an Amicon ultra device (10 000 Da, Millipore) by centrifugation at 3000 × g for 5 min. The drug amount was determined by a high-performance liquid chromatography (HPLC) system (Waters Alliance, USA) on an Eclipse XDB-C18 column (4.6 × 150 mm, Agilent, USA) at room temperature. The mobile phase consisted of water (pH 4.0)−acetonitrile (60:40, v/v) with the flow rate of 1.0 mL/ min, and the confluent was detected at 235 nm. The in vitro release profiles of SA-TSN in phosphatebuffered solution (PBS) at different pH values (5.0, 6.0, and 7.0) were measured at 37 °C by a dialysis method. Samples were collected at predetermined time intervals and analyzed by the aforementioned HPLC method. In Vitro Antiproliferative Activity. The antiproliferative activity of SA-TSN was measured in sensitive MCF-7 and resistant MCF-7/ADR cells to evaluate the in vitro efficacy in reducing MDR. Cells were seeded into a 96-well plate at 8 × 103 cells/well and maintained in the culture media for 24 h. Then, fresh media with series concentration of teniposide solution (TS), TSN, and SA-TSN was added to the wells and incubated for further 48 h. Cells treated with blank culture media was performed as a negative control. Afterward, the cytotoxicity was measured by MTT assay by recording the absorbance values at 570 nm on a microplate reader (Bio-Rad model 550, USA). The cell viability was calculated as the ratio between the absorbance values of these samples and that of negative control. Meanwhile, the half-maximal inhibitory concentration (IC50) of each group was calculated by the GraphPad Prism 5 software. The resistance index of each group was defined as the ratio between the IC50 value in resistant MCF-7/ADR cells and that in sensitive MCF-7 cells. Mechanism of Reduced MDR by SA-TSN. Cellular Uptake. Sensitive MCF-7 cells and resistant MCF-7/ADR cells were seeded into 24-well plates at 2 × 105 cells/well and cultured overnight prior to the experiments. TS, TSN, or SATSN was added to each well at 5.0 μg/mL of teniposide and incubated for 2.0 h. Then, the culture media were removed, and cells were rinsed with cold PBS (pH 7.4). Afterward, 100 μL of water was added to each well, and cells were scrapped. The cell suspension was collected, mixed with 300 μL of acetonitrile, homogenized by sonication for 10 s, and centrifuged at 10 000 × g for 10 min. The drug amount in the supernatant was determined by HPLC methods as described above.

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EXPERIMENTAL SECTION Materials. Stearylamine was provided by Aladdin Reagent Co. Ltd. (Shanghai, China). Teniposide (99.2%) was purchased from Beijing Chemsynlab Pharmaceutical Science & Technology Co. Ltd. (Beijing, China). Solutol HS-15 was obtained from BASF Co. Ltd. (Ludwigshafen, Germany). D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) was supplied by Sigma-Aldrich (MO, USA). Medium chain triglyceride (MCT) was provided by Tieling Beiya Medicinal Oil Co. Ltd. (Liaoning, China). Acetonitrile for HPLC measurement was obtained from Merck Co. Ltd. Other chemicals and reagents were supplied by the Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Cell Culture. Human breast cancer cell line (MCF-7) was obtained from Shanghai Cell Resource Center of Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (CAS). Doxorubicin-resistant MCF-7/ADR cells were obtained from the KeyGEN Biotech (Nanjing, China). Cells were cultured in RPMI 1640 culture media (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Biochrom, Germany), 100 units/mL penicillin G sodium, and 100 μg/mL streptomycin sulfate. To maintain the drug resistant phenotype of MCF-7/ADR, cells were maintained in the culture media with 1.0 μg/mL of doxorubicin. The cell culture was performed at 37 °C in a humidified and 5% CO2 incubator for further experiments. Animals. Sprague−Dawley (SD) rats (200−220 g, ♂) and BALB/C nude mice (18−22 g, ♀) were provided by Shanghai Experiment Animal Center, CAS. Animals were kept under the animal care facility and acclimatized for 5 days prior to the experiment. The in vivo experiments were carried out according to the protocols approved by the International Animal Care and Use Committee (IACUC) of Shanghai Institute of Materia Medica, CAS. Preparation and Characterization of SA-TSN. The teniposide loaded self-assembled nanocarrier based on SA (SA-TSN) was prepared as our previously described method.31 Briefly, teniposide, SA, TPGS, MCT, and HS-15 (1:1:20:15:10, w/w) was accurately weighted and dissolved in acetone in a round flask. The mixed solution was evaporated to dryness under reduced pressure (Heidolph Laborata 4000, Germany) to form a thin film. Then, the film was dispersed in doubledistilled water at 37 °C by gentle shaking, and SA-TSN could be spontaneously formed due to the hydrophobic interaction among the involved ingredients. As a control, a teniposide B



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Detection kit (Invitrogen, USA) using as a flow cytometer (BD, USA). Cells were seeded into 6-well plates at 3 × 105 cells/well in 2.0 mL of culture media. After 24 h, TS, SN, TSN, SA-SN, and SA-TSN were added to each well at 2.0 μg/mL of teniposide and incubated for further 48 h. Then, cells were respectively stained with the Annexin V-FITC and propidium iodide (PI, provided in the kit) and analyzed using the FACSCalibur flow cytometry system (Becton Dickinson, USA). By contrast, cells without any treatment were performed as a negative control. In Vivo Pharmacokinetic Behavior in Rats. The pharmacokinetic behavior of SA-TSN and TSN was investigated in rats in comparison with that of TS. Prior to the experiments, rats were fasted overnight with free access to water and divided into three groups (n = 4). SA-TSN, TSN, and TS were intravenously administered to rats at 10 mg/kg of teniposide. Samples were collected into heparinized tubes at certain time intervals, and plasma was immediately separated by centrifugation at 10 000 × g for 5 min. Then, 25 μL of plasma samples were mixed with 25 μL of berberine (10.0 ng/mL, internal standard) and 125 μL of acetonitrile by vortex for 1.0 min, and centrifuged at 12 000 × g for 5 min to precipitate the plasma proteins. The teniposide concentration was determined by LC-MS/MS analysis. The chromatographic separation was performed on LC30AD system (Shimadzu, Japan) with the following conditions: Gemini C18 column (50 × 2.0 mm I.D., 5 μm, Phenomenex, USA) with a security guard C18 column (Phenomenex, USA); the temperature is 40 °C with a flow rate of 0.6 mL/min. The mobile phase consisted of with a gradient mixture of acetonitrile (solvent A) and 5 mM of ammonium acetate with 0.1% (v/v) formic acid (solvent B) with the following gradient program: 0−0.5 min: 20% A; 0.5−1.5 min: from 20% to 70% A; 1.5−2.3 min: 70% A; 2.3−3.2 min: from 70% to 20% A and followed by re-equilibration at 20% A until 4.0 min. The mass spectrometer (Qtrap 5500, Applied Biosystems, USA) was operated in positive mode, and the operational parameters of ESI source were the following: vaporizing temperature, 350 °C; declustering potential (DP), 120 V; nitrogen sheath gas, 55 psi; nitrogen curtain gas, 30 psi; capillary potential 3500 V; dwell time of 200 ms. Detection and quantification were monitored using multiple reaction monitoring (MRM) with the following transitions: m/z 674.2 → m/z 383.2 for teniposide and m/z 366.1 → m/z 292.1 for berberine with the optimized collision energy (CE) was 32 and 39 eV, respectively. The pharmacokinetic parameters were calculated by noncompartmental model with DAS 2.1.1 software. In Vivo Distribution of SA-TSN in Resistant Breast Tumor. The resistant breast tumor was induced in BALB/C nude mice by subcutaneous injection of resistant MCF-7/ADR cells. Briefly, approximately 100 μL of cell suspension (4 × 106 cells) was subcutaneously injected into the right flanks of BALB/C nude mice. The nude mice were maintained in the animal care facility until the tumor volumes reached 80−100 mm3 for further experiments. The in vivo distribution of SA-TSN was visualized under the in vivo imaging system (Carestream FX Pro, USA) and LCSM (Fluoview FV 1000, Olympus, Japan). SA-TSN was fluorescently labeled with hydrophobic Nile red in the same preparation procedure. The fluorescently labeled SA-TSN was injected into mice at 10 mg/kg of teniposide and 3 mg/kg of Nile red. Mice were sacrificed at 0.5 h after intravenously administration, and various typical organs were removed. These

P-gp Expression in MCF-7/ADR Cells. MCF-7/ADR cells were seeded into 24-well plates at 5 × 104 cells/well and cultured for 24 h for the attachment. TS, SN, TSN, SA-SN, and SA-TSN were added to the culture media at 2.0 μg/mL of teniposide and incubated for further 48 h. Then, cells were trypsinized with trypsin-EDTA solution and collected with centrifugation at 2000 × g for 1.5 min. Afterward, the cell pellets were rinsed with cold PBS (pH 7.4) and resuspended in PBS for further analysis. The P-gp on cell surface was labeled with phycoerythrin (PE)-antihuman MDR1 (CD243, P-gp, ABCB1, eBioscience, USA) while the nonspecific labeling was corrected by PE-Mouse IgG2a (eBioscience, USA). Then, the P-gp expression were measured with flow cytometry (Becton Dickinson, USA) by monitoring the fluorescent intensity and analyzed with the CellQuest software. Cell Cycle Analysis. MCF-7/ADR cells were seeded into 6well plates at a density of 3 × 105 cells/well and cultured overnight. TS, SN, TSN, SA-SN, and SA-TSN were added to each well at 2.0 μg/mL of teniposide and incubated for further 48 h. Then, cells were harvested, rinsed, and resuspended in 300 μL of PBS, fixed with 700 μL of cold ethyl ethanol, and stored at 4 °C for 24 h. After centrifugation at 2500 × g for 5 min, the cell pellets were rinsed, resuspended in 500 μL of PBS (pH 7.4), and incubated with RNase A (40 μg/mL) for 60 min at 37 °C. Thereafter, cells were stained with 10 μg/mL of propidium iodide (PI, Sigma) for 30 min in the dark and analyzed by the FACScan flow cytometry system (Becton Dickinson, France). The percentage of cells in each cell cycle was calculated by the ModFit software (Verity Software House, ME). Co-localization of SA-TSN in MCF-7/ADR Cells. The subcellular localization of SA-TSN in resistant MCF-7/ADR cells was observed under laser confocal scanning microscopy (LCSM, Fluoview FV 1000, Olympus, Japan). Cells were grown on the round glass coverslips (Ø10 mm) at 5 × 104 cells/well in a 24-well culture plate (Corning, USA) and cultured overnight. SA-TSN was fluorescently labeled with hydrophobic Nile red (Red, Acros) and incubated with MCF7/ADR cells at 37 °C for 4 h. The lysosome was labeled with LysoTracker Green DND-26 (Green, Molecular probes) to investigate its subcellular localization in lysosome. Similarly, cells were stained with Mitotracker Green (Green, Molecular probes) to determine the localization of SA-TSN in mitochondria. The nuclei were counterstained with Hoechst 33342 (Blue, Sigma). Thereafter, the coverslips were rinsed with phenol-red free RPMI 1640 culture media, mounted on the glass microscope slides with antifade solution, and visualized under the LCSM. Mitochondrial Transmembrane Potential (Δψm) Measurement. MCF-7/ADR cells were seeded into 12-well plated at a density of 2 × 105 cells/well and cultured overnight. TS, SN, TSN, SA-SN, and SA-TSN were added to each well at 2.0 μg/ mL of teniposide and incubated for 24 h. Cells were rinsed with PBS (pH 7.4), harvested with trypsin-EDTA solution and collected with centrifugation at 2, 000 × g for 2 min. Then, the cell pellets were stained with the JC-1 mitochondrial membrane potential assay kit (Beyotime, China) according to the manufacturer’s protocol. Mitochondria depolarization was characterized by a switch from the red to green fluorescence intensity. The fluorescent intensity of each sample was detected using a FACScan flow cytometer (BD, USA). Cell Apoptosis. The apoptosis of MCF-7/ADR cells was determined by staining with the Annexin V-FITC Apoptosis C



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Figure 1. Characterization of SA-TSN. (A) Typical TEM images of SA-TSN (scale bar = 100 nm); (B) size distribution of SA-TSN characterized by intensity; (C) X-ray diffraction patterns of teniposide, stearylamine (SA), TSN, and SA-TSN; (D) in vitro release profiles of teniposide from SATSN in various release media at different pH values.

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RESULTS AND DISCUSSION Physicochemical Characterization of SA-TSN. SA was selected for the preparation of SA-TSN to reduce the expression of P-gp and enhance the targeted delivery of teniposide to mitochondria for reversal of MDR. The experimental results indicated that SA-TSN exhibited nanometer-sized spherical particles under TEM microscope (Figure 1A). SA-TSN showed the mean diameter of 31.59 ± 3.43 nm with the polydispersity index of 0.207 ± 0.024 (Figure 1B), which was in accordance with that of TEM measurements. The encapsulation efficiency of teniposide in SA-TSN was 99.25 ± 0.21%, which indicated that the lipophilic teniposide was entirely entrapped in the nanocarrier. The crystalline state of teniposide in SA-TSN was determined by XRD analysis (Figure 1C). Many characteristic crystalline diffraction peaks were observed in the profiles of teniposide and SA. However, these diffraction peaks disappeared in the profile of SA-TSN, which implicated that teniposide and SA could present as amorphous or molecular state (Figure 1C). The in vitro release of teniposide from SA-TSN showed a burst release within 1.0 h and then continuously increased with time (Figure 1D). Moreover, the in vitro release rate and extent of teniposide from SA-TSN within 8.0 h was changed with the pH value of release media, which was the lowest at pH 7.0 and then slightly increased at pH 5.0 and 6.0. Since the aqueous solubility of teniposide was not obviously changed at these different pH values, the higher release kinetic of teniposide from SA-TSN at lower pH values could result from the effect of weak acidic solution on the structure of cationic SA-TSN. Due to the low pH microenvironment of tumor tissue, the varied release profiles of SA-TSN with the pH values of release media could be advantageous for cancer therapy.10 In Vitro Efficacy and Possible Mechanism. The in vitro antiproliferation activity of SA-TSN was evaluated in sensitive MCF-7 and resistant MCF-7/ADR cells. In MCF-7 cells, the cytotoxicity of SA-TSN was similar with that of TSN, which

organs were rinsed with cold physiological saline and then visualized under the in vivo imaging system (Carestream FX Pro, USA). Similarly, after the removal of these organs, tissues were rinsed and frozen in embedding media (OCT, SAKURA, Japan) for subsequent cryostat sectioning at 20 μm (Mev, SLEE, Germany). The sections were applied to glass slides, washed with cold PBS, and fixed with 3.7% buffered formalin for 10 min at room temperature. Afterward, the sections were stained with phalloidin-FITC (Green, Sigma) and DAPI (Blue, Sigma) according to the manufacturer’s protocols for subsequent observation under LCSM (Olympus, Japan). Tumor Inhibitory Effect of SA-TSN in Resistant Breast Tumor. The in vivo inhibitory efficacy of SA-TSN was evaluated in MCF-7/ADR cells induced resistant xenograft model. When tumors reached 80−100 mm3, animals were randomly divided into four groups (n = 6) and intravenously administered with saline, TS (10 mg/kg), TSN, or SA-TSN (10 mg/kg of teniposide), respectively. Mice were administered every 4 days over a period of 20 days. Meanwhile, the body weight, length, and width of tumor were measured every 4 days to monitor the tumor progression. The tumor volume was calculated as the following: V = [length × (width)2]/2; relative tumor volume = tumor volume/primary tumor volume. At day 20 after administration, mice were sacrificed, and tumors from each group were removed, rinsed, accurately weighed, and photographed. The tumor inhibitory rate (TIR) was calculated from the formula: TIR = 100% − Wtest/Wsaline × 100%. Therein, Wtest was the average tumor weight of tested groups (TS, TSN, or SA-TSN groups), while Wsaline referred to that of saline group. Statistical Analysis. Data were expressed as mean ± SD and statistically analyzed using the one way analysis of variance (ANOVA). A significant difference was considered as p < 0.05, and very significance was regarded as p < 0.01. D



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Figure 2. In vitro evaluation of SA-TSN on reducing the MDR of MCF-7/ADR cells. (A) Cell viability of SA-TSN, TSN, and teniposide solution (TS) in sensitive MCF-7 cells; (B) cell viability of SA-TSN, TSN, and TS in MCF-7/ADR cells; (C) IC50 values SA-TSN, TSN, and TS in MCF-7 and MCF-7/ADR cells (** p < 0.01); (D) cellular uptake of SA-TSN, TSN, and TS in MCF-7 and MCF-7/ADR cells (*p < 0.05); (E) cell cycle analysis of MCF-7/ADR cells treated with SA-TSN, SA contained self-assembled nanocarrier (SA-SN), TSN, self-assembled nanocarrier without SA and teniposide (SN), and TS (*p < 0.05; ** p < 0.01); (F) P-gp expression in MCF-7/ADR cells treated with SA-TSN, SA-SN, TSN, SN, and TS; (G) Subcellular localization of SA-TSN with lysosome in MCF-7/ADR cells; (H) subcellular localization of SA-TSN with mitochondria in MCF-7/ ADR cells; (I) mitochondria membrane potential values in MCF-7/ADR cells treated with SA-TSN, SA-SN, TSN, SN, and TS; (*p < 0.05; **p < 0.01); (J) cell apoptosis of MCF-7/ADR cells treated with SA-TSN, SA-SN, TSN, SN, and TS.

to 3.69 μg/mL with the resistant index was only 1.52 (Figure 2C). Thereby, the MDR of teniposide in MCF-7/ADR cells could be effectively reduced by SA-TSN, which could mainly attribute to the cooperative combination of SA and other involved ingredients in SA-TSN. Subsequently, several experiments associated with P-gp efflux pumps and mitochondria were performed to elucidate the possible mechanism of reduced MDR by SA-TSN. First, the cellular uptake in MCF-7 and MCF-7/ADR was quantified by HPLC analysis (Figure 2D). The experimental results indicated the teniposide concentration in resistant MCF-7/ADR cells

was higher than that of TS (Figure 2A). However, in resistant MCF-7/ADR cells, the cytotoxicity of SA-TSN was significantly higher than that of TSN and TS (Figure 2B). The IC50 values of teniposide in MCF-7 and MCF-7/ADR cells were 5.43 and 59.91 μg/mL, respectively, and the resistant index was 11.03, which confirmed the severe resistance of teniposide in MCF-7/ ADR cells (Figure 2C). The IC50 value of TSN in MCF-7/ADR cells was 35.83 μg/mL with the resistant index of 10.73, which indicated the drug resistance of teniposide could not be reduced by TSN. However, when MCF-7/ADR cells were treated with SA-TSN, the IC50 value was significantly reduced E



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Figure 3. In vivo behavior of SA-TSN. (A) Pharmacokinetic profiles of intravenously administrated SA-TSN, TSN ,and TS in rats (10 mg/kg). (B) The ex vivo imaging of Nile red labeled SA-TSN in MCF-7/ADR cells induced xenografts. (C) LSCM images of fluorescent SA-TSN in different organs of MCF-7/ADR cells induced xenografts.

7/ADR cells was not evidently changed in the presence of SN but obviously reduced about 90% with the treatment of SA-SN (Figure 2F). Moreover, statistical significance occurred between the P-gp expression in MCF-7/ADR cells treated with TSN and SA-TSN, but no significance was detected between that of SASN and SA-TSN. Thereby, the notable inhibition of P-gp expression in MCF-7/ADR cells by SA-TSN could attribute to the incorporation of SA into the nanocarrier, which could be one of the major mechanisms for the reversal of MDR. Mitochondria played key roles in mediating intrinsic pathway of cell death by apoptosis and emerged as targets for cancer therapy.11−13 Moreover, the mitochondrial targeted drug delivery could be promising to induce apoptosis in resistant cancer cells.11,15 Herein, the subcellular localization of SA-TSN in resistant MCF-7/ADR cells was observed under LCSM. SATSN was fluorescently labeled with hydrophobic Nile red, while the lysosomes and mitochondria was respectively labeled with green fluorescent probes. The bright yellow fluorescence color (a composition of red and green) was used to indicate the colocalization of fluorescent SA-TSN with lysosome or mitochondria. The captured imaged showed a few yellow spots in lysosomes of MCF-7/ADR cells (Figure 2G), but plenty of yellow fluorescence in mitochondria of MCF-7/ADR cells (Figure 2H). Thereby, SA-TSN could be selectively accumulated into the mitochondria of resistant MCF-7/ADR cells, which could result from the mitochondrial targeting characters of the hydrophobic part of TPGS and SA included in SA-TSN.12,23 Moreover, the mitochondrial membrane potential (Δψm) was an important parameter of mitochondria function, and the loss of Δψm was usually considered as a hallmark of apoptosis.33,34 Compared with the negative control, the Δψm value of MCF-7/ADR cells was not significantly changed with the incubation of SN, but evidently reduced about 70% after the treatment of SA-SN, which could be ascribed to the

treated with TS, TSN, and SA-TSN was much lower than that in sensitive MCF-7 cells, which indicated that the reduced cellular uptake of teniposide could be an important reason for the drug resistance. However, no significant difference was detected among the cellular uptake of TS, TSN, and SA-TSN in MCF-7 cells. Second, the cell cycles of MCF-7/ADR cells treated with TS, TSN and SA-TSN were analyzed. Compared with the negative control, the cell cycle distribution percentages in each phase in MCF-7/ADR cells were not significantly changed when cells were treated with TS, SN, and SA-SN (Figure 2E). However, after the treatment of TSN and SATSN, the cell cycle percentages in G2 phase was increased from 13.15% to 59.45% and 50.78%, respectively, and that in G1 phase was significantly reduced from 52.82% to 12.31% and 14.68%. Thereby, the cell cycle of MCF-7/ADR cells at each phase was not significantly changed with the treatment of blank nanocarrier (SN or SA-SN) but mainly arrested in G2 phase after the treatment of TSN and SA-TSN. Since teniposide could block the cell cycle in S-phase and G2 phase to induce the apoptosis in tumor cells, the arrestment of MCF-7/ADR cells in G2 phase induced by TSN and SA-TSN could be potential to exert its antiproliferation activity.32 Third, since the P-gp efflux pumps was overexpressed on the surface of resistant MCF-7/ADR cells, the P-gp expression was determined by a flow cytometer. Since SA was almost insoluble in water, the effect of on the P-gp expression was performed after its incorporation into self-assembled nanocarrier without teniposide (SA-SN). Compared with the negative control, the P-gp content in MCF-7/ADR cells was not significantly changed in the presence of TS but significantly reduced about 30% with the treatment of TSN and greatly decreased over 90% when cells were treated with SA-TSN, which indicated that the P-gp expression on the surface of MCF-7/ADR cells was greatly inhibited by SA-TSN. Meanwhile, the P-gp expression in MCFF



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Figure 4. In vivo efficacy of SA-TSN on reducing the MDR in resistant MCF-7/ADR induced xenografts. (A) The tumor growth inhibitory profiles in resistant MCF-7/ADR induced xenografts treated with saline, teniposide solution (TS), TSN, and SA-TSN (10 mg/kg). (B) The body weight changes in resistant MCF-7/ADR induced xenografts during the treatment. (C) The photographs of tumors from each group excised on day 20. (D) The growth inhibitory rate of TS, TSN, and SA-TSN in resistant MCF-7/ADR induced xenografts in comparison with that of saline group (*p < 0.05).

effective incorporation of SA into SN. Moreover, the Δψm of MCF-7/ADR cells was respectively decreased about 44% and 35% after the treatment of TS and TSN and greatly reduced to 10% when cells were incubated with SA-TSN. Thereby, the obvious reduction of Δψm value by SA-TSN could be result from the effective combination of teniposide and SA-SN, which could be promising for inducing the apoptosis of resistant MCF-7/ADR cells. Furthermore, the induced apoptosis of resistant MCF-7/ADR cells by SA-TSN was characterized by counting the apoptotic percentages of the early and late periods (Figure 2J). When MCF-7/ADR cells was treated with TS, TSN, and SA-TSN at 2.0 μg/mL of teniposide, the induced apoptotic percentages was 0.98%, 6.47%, and 92.64%, respectively, which indicated that the cell apoptosis in resistant MCF-7/ADR cells could be significantly enhanced by SA-TSN. In addition, SA-SN induced a 2.28% of cell apoptosis and 35.43% of cell death, which could be favorable for the reversal of MDR. These experimental results certified that the MDR in resistant MCF-7/ADR could be greatly reduced by SA-TSN. The mitochondria targeted delivery of teniposide could be another important mechanism for reversal of MDR by SA-TSN. In Vivo Behavior of SA-TSN. The pharmacokinetic behavior showed that a significant difference occurred between the plasma drug concentration−time profiles of TS and that of SA-TSN or TSN (Figure 3A) but was not detected between the profiles of SA-TSN and TSN. At each time point after intravenous administration, the teniposide concentration in rats treated with SA-TSN or TSN was obviously higher than that of TS. Similarly, the area under concentration−time curve (AUC) of SA-TSN (20231.6 ± 478.2 ng·h/mL) and TSN (21165.4 ± 3145.1 ng·h/mL) was respectively 16.79 and 17.57-fold higher than that of TS (1204.8 ± 277.3 ng·h/mL). The significant difference between in vivo pharmacokinetic behavior of SATSN and that of TS could attribute to the incorporation of lipophilic teniposide into SA-TSN with high encapsulation efficiency, which could beneficial to increasing the systemic circulation time of teniposide and enhancing its distribution in the tumor site for further cancer treatment.

The in vivo biodistribution of SA-TSN was measured in resistant MCF-7/ADR induced xenografts. SA-TSN was fluorescently labeled with the hydrophobic nile red and then intravenously administered to the tumor-bearing mice. The ex vivo photographs indicated that the fluorescent SA-TSN was mainly distributed in tumor, liver, lung, and kidney, but lowest in spleen (Figure 3B). Then, these tissues were sectioned for further observation under LCSM. The nuclei were stained with DAPI (Blue). The LCSM images indicated that plenty of red fluorescence was detected in liver, lung, and tumor, which was in accordance with that of ex vivo observation (Figure 3C). In particular, in the tumor site, a large amount of red spots was visualized on the surface of tumor site, and the fluorescent intensity was reduced from the surface to the inner site (Figure 3C). The reason could attribute to the physiological properties of solid tumor, which was surrounded by abundant blood vessels around the tumor. In addition, a few red spots were observed in the inner site of tumor site, which could result from the nanometeric size of SA-TSN. It has been reported that only 30 nm micelles could penetrate poorly permeable pancreatic tumors to achieve an antitumor effect.35 The enhanced accumulation of SA-TSN in tumor and its penetration into the interior of tumor could be beneficial to enhancing the antitumor activity of teniposide. In Vivo Inhibitory Effect of SA-TSN in MCF-7/ADR Induced Xenografts. The in vivo antitumor effects of SATSN in resistant MCF-7/ADR induced xenografts were performed in comparison with that of TSN and TS. The tumor volume and body weight of tumor-bearing mice were monitored every 4 days within 20 days. In the tumor growth profiles, the tumor volumes in mice treated with SA-TSN were much lower than that with the treatment of saline, TS or TSN. Compared with the saline group, the tumor volume at day 20 after treatment was 59.60 ± 21.47% for TS, 44.88 ± 7.73% for TSN, and 21.57 ± 8.84% for SA-TSN, which indicated that SATSN achieved superior tumor growth inhibitory efficacy than TS and TSN (Figure 4A). In addition, no significant difference was detected among the body weight of tumor-bearing mice G



Article

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treated with TS, TSN, and SA-TSN, and no animals were dead during the treatment, which confirmed the good biosafety of SA-TSN in tumor therapy (Figure 4B). At the end point of day 20 after treatment, mice were euthanatized, and tumors were removed and photographed (Figure 4C). Then, tumors were weighed, and the tumor inhibitory rates were calculated. Compared with the saline group, the tumor inhibitory rate of SA-TSN in resistant MCF-7/ADR induced xenografts was 76.48 ± 8.27%, which was significantly higher than that of TS (42.63 ± 11.40%) and TSN (57.97 ± 3.91%) (Figure 4D). Thereby, SA-TSN could be more effective to inhibit the tumor growth in resistant MCF-7/ADR induced xenografts and reduce MDR.

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CONCLUSION The MDR of resistant MCF-7/ADR cells was greatly reduced by SA-TSN, which could attribute to the markedly reduced expression of P-gp, increased percentages in G2 phase, selectively accumulation in mitochondria, decrease of mitochondrial membrane potential, and greatly improved apoptosis. The plasma concentration of teniposide was greatly improved by SA-TSN, and the intravenously administered SA-TSN could accumulate in the tumor site and penetrate into the inner site of tumor in MCF-7/ADR induced xenografts. In particular, SATSN exhibited superior antitumor activity in MCF-7/ADR induced models than TSN and TS, which verified the effectiveness of SA-TSN in reversal of MDR. Thereby, SATSN could be potential to circumvent MDR in chemotherapy.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Y.L.) or [email protected] (H.Y.); 501 Haike Road, Shanghai 201203, China; Tel./Fax: +86-21-2013-1979. Author Contributions

Z.Z. and Z.L. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The National Basic Research Program of China (2010CB934000, 2012CB932502, 2013CB932503, and 2013CB932704), the National Natural Science Foundation of China (30925041, 81270047), and Shanghai Program (11 nm0505900) are gratefully acknowledged for financial support.

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Reversal of Multidrug Resistance by Mitochondrial Targeted Self

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