Mitoxantrone- and Folate-TPGS2k Conjugate Hybrid Micellar

Feb 13, 2017 - Mitoxantrone (MTO) is a potent drug used to treat breast cancer; however, efforts to expand its clinical applicability have been restri...
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Mitoxantrone- and Folate-TPGS2k Conjugate Hybrid Micellar Aggregates To Circumvent Toxicity and Enhance Efficiency for Breast Cancer Therapy Nida El Islem Guissi,†,‡ Huipeng Li,† Yurui Xu,† Farouk Semcheddine,§ Minglei Chen,† Zhigui Su,*,† and Qineng Ping*,† †

State Key Laboratory of Natural Medicines, Department of Pharmaceutics, and Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China Pharmaceutical University, Nanjing 210009, China ‡ Department of Pharmacy, Faculty of Medicine, Ferhat Abbas University, Setif 19000, Algeria § State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China S Supporting Information *

ABSTRACT: Mitoxantrone (MTO) is a potent drug used to treat breast cancer; however, efforts to expand its clinical applicability have been restricted because of its high risk for cardiotoxicity. In this study, we successfully conjugated MTO or folic acid (FA) to a synthesized D-α-tocopheryl polyethylene glycol 2000 succinate (TPGS2k), herein, shortened to MCT and FCT, respectively. The two produced conjugates could self-assemble to form MCT micelles or MCT/FCT mixed micelles (FMCT) aiming to lower systemic toxicity, enhance entrapment efficiency, and provide a platform for targeted delivery. Moreover, these micellar materials showed a significantly low CMC and could be used to load MTO. The diameters of MTO-loaded micelles (MTO-MCT and MTO-FMCT) were less than 100 nm with a negative zeta potential. We further characterized the pH-responsive drug release of MTO-MCT and MTO-FMCT and then assessed their cellular uptake and antitumor efficacy in human breast cancer cell lines (MCF-7) via confocal microscopy, flow cytometry, and cytotoxicity studies. All the results revealed that both MTO-MCT and MTO-FMCT increased drug loading and entrapment efficiency and possessed sufficient pH-sensitive release. Additionally, MTO-FMCT displayed an improved uptake through folatemediated endocytosis, resulting in a higher cytotoxic effect on MCF-7 cells compared with that of MTO-MCT. Meanwhile, both MTO-MCT and MTO-FMCT exhibited a low toxicity on hCMEC/D3 normal cells. More importantly, pharmacokinetic study demonstrated that, in comparison with free MTO injection, MTO-MCT and MTO-FMCT, respectively, achieved half-lives 11.5 and 13 times longer and a 9.7- and 5.8-fold increase in AUC. In vivo, both MTO-MCT and MTO-FMCT formulations significantly prolonged the survival time of MCF-7 tumor-bearing mice and had a better efficacy/toxicity ratio. Promisingly, MTO-FMCT micelles remarkably increased MTO accumulation in tumors in vivo, induced higher tumor cell apoptosis, and showed lower toxicity toward major organs. These results imply that MTO-FMCT may be used as a potential drug delivery system for breast cancer targeted therapy. KEYWORDS: mitoxantrone, folate targeting, TPGS2k, self-assembled micelle, breast cancer therapy

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INTRODUCTION

reported as being able to cross biological barriers and deliver anticancer cargos to their specific recipients.5 Among the various drug delivery systems, amphiphilic copolymeric micelles have gained popular consideration in nanomedicine research for their various desirable features. Those physiologically friendly building blocks of copolymers are used to design micelles with prolonged half-lives and

Breast cancer occupies the second place, after lung cancer, on the list of cancers with the highest mortality rates among women.1 Chemotherapy, which is a powerful tool in breast cancer treatment, has toxic effects on both cancerous and normal cells leading to multiple side effects such as heart failure, bone marrow suppression as well as organ (i.e., kidney, lung, liver) damage. Therefore, finding the fusion between treatment potency and patient tolerance is essential.2−4 In order to overcome these side effects, nanotechnology provides multifaceted solutions. Indeed, nanocarriers are © 2017 American Chemical Society

Received: Revised: Accepted: Published: 1082

November 7, 2016 February 8, 2017 February 13, 2017 February 13, 2017 DOI: 10.1021/acs.molpharmaceut.6b01009 Mol. Pharmaceutics 2017, 14, 1082−1094

Article

Molecular Pharmaceutics

pH-sensitive micellar material involving the conjugation of MTO to a newly synthesized TPGS2k to reduce or overcome MTO’s undesirable toxicity and achieve a synergetic anticancer effect. First, we successfully synthesized TPGS2k by conjugating a bis-amine PEG2k to d-α-tocopheryl succinate (α-TOS). PEG2k with two terminal amino groups was chosen so that one terminal would react with the carboxyl group in α-TOS to form TPGS2k, whereas the second amino group would be kept to obtain TPGS derivatives.9 Second, on one hand, TPGS2k was conjugated to MTO through amidation, which was previously reacted with succinic anhydride (SA) to obtain MTO disuccinic ester (MTO-SA). On the other hand, a fraction of TPGS2k was reacted with FA to obtain FCT, aiming to target the FR expressed on the tumor cells surface, thus allowing an enhanced drug release control at the tumor site and an improved cellular uptake of chemotherapeutic drugs. Finally, MTO-loaded MCT micelles (MTO-MCT) and MTO-loaded FCT/MCT mixed micelles (MTO-FMCT) were prepared on the basis of earlier reports asserting that high drug loading capacity could be achieved if the chemical structure of the drug was similar to that of polymer micelles. This mechanism is considerably comparable to the “like dissolves like” rule, even though the core of polymeric micelles does not actually dissolve MTO.23 In our study, we evaluated, in vitro, the physicochemical properties, pH-responsive drug release, cellular uptake, and antitumor efficacy of MTO-MCT and MTO-FMCT. We illustrated the efficiency in vivo, by investigating their prolonged action, their pharmacodynamics, and their safety. To the best of our knowledge, MTO has never been conjugated to a TPGS2k polymer for both cancer-cell-targeting ability and MTO undesirable toxic effects diminution purposes (Scheme 1).

improved control of drug release within the body. Recently, micellar nanocarriers containing a drug-release-responding mechanism with specific targeting have been constructed.6 The pH-responsive polymers have been valued as the most compelling candidates due to their stability under physiological conditions (blood, pH = 7.4) and disassembly under acidic environment (endo/lysosome, pH = 5.0), hence triggering drug release.6 Those properties would lead to a notably increased anticancer efficacy, higher tolerance, and lesser drug resistance.6−8 In addition to their technical simplicity, copolymeric micelles present some advantages over other delivery systems by displaying a superior biocompatibility and drug delivery efficacy.9 D-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS1k), which possesses an amphiphilic structure, has been reported to have anticancer activities and to be able to overcome multidrug resistance (MDR). Recent studies have demonstrated that TPGS mixed micelles can improve drug permeability and cellular uptake, resulting in a higher therapeutic efficacy.6,7 Moreover, polymeric micelles might benefit from PEG chain length in TPGS2k to avoid being captured and disposed of by the cells of reticuloendothelial system,9 which results in a longer circulation lifetime.10 In our work, we chose the synthesized TPGS2k (with PEG2k bisamine) over TPGS1k not only because of its ability to assemble into stabilized micelles but also because of (1) the relative stability of the amide function compared to ester function when it comes to hydrolysis in blood. (2) When subjected to the weak acidic environment of the extracellular fluid surrounding cancer cells, amides would hydrolyze and restore cationic amines. Furthermore, the amide hydrolysis rate could be adjusted by selecting a suitable anhydride;11 (3) intracellular drug release would occur through endolysosomal pH-sensitive linkers such as cis-aconityl, hydrazine, or amide bonds.12,13 Furthermore, targeting devices such as peptides, aptamers, or folate, can be synthetically linked to the PEG termini by activating its reactive functional groups, which would specifically bind to tumor cells while exhibiting no affinity for healthy cells. Among these targeting agents, folic acid (FA) was proven to be a promising targeting moiety for its low to no expression on normal cells. Additionally, the structure of FA bound to folate receptor (FR) has recently been elucidated. The finding demonstrated that FA, after its conjugation to drugs via its γ-carboxylic group, preserves its binding characteristics to FR.14,15 Mitoxantrone (MTO), an anthracycline, is among the most active agents against breast cancer. However, its risk of cardiotoxicity limited its applicability. MTO cytotoxicity has been attributed, at least in part, to its double side chain hydroxyl groups. Indeed, these hydroxyethylaminoalkyl side chains were found to be partly responsible for the DNA recognition and its binding to MTO,16−18 and also, the oxidation of these terminal hydroxyl groups would engender the formation of MTO metabolites (i.e., monocarboxylic and dicarboxylic acids).19 Those metabolites, which were found to be toxic, highlight the importance of MTO metabolism in the emergence of harmful effects.20 Thus, a strategy to overcome the side effect of MTO is necessary to expand its application. Knowing that TPGS1k-doxorubicin conjugate improved the drug’s therapeutic effect,21 that FA conjugation enhanced the cellular uptake of docetaxel-loaded TPGS micelles,9 and that a drug-TPGS conjugation might lead to a synergetic effect,22 we, inspired by the above-mentioned reports, developed a novel

Scheme 1. Schematic Diagram of MTO-FMCT Formation and Schematic Illustration of MTO-FMCT Targeting Delivery to Tumor and Tumor Cella

a

I: the folate-receptor-mediated endocytosis; II: the degradation of MTO-FMCT in the acidic microenvironment of endo/lysosome following MTO release; III: the accumulation of MTO into the nucleus; IV: MTO-induced cell death.

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MATERIALS AND METHODS Materials. MTO was purchased from Sichuan Pharmaceutical Co. Ltd., China. D-α-Tocopheryl succinate was provided by Hebei Baiwei Biotechnology Co., Ltd. Bis amine polyoxyethylene (MW 2000 Da), folic acid, 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC), 4-dimethy1083

DOI: 10.1021/acs.molpharmaceut.6b01009 Mol. Pharmaceutics 2017, 14, 1082−1094

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Molecular Pharmaceutics

Micelles Preparation. Micelles have been prepared on the basis of the method used by Mi Yu et al.9 After MTO and coumarin-6 (C6) were weighed and dissolved in chloroform, the two solutions were then separately mixed with a chloroform solution of MCT. Rotary vacuum evaporation was used to eliminate the organic solvent. After freeze-drying the obtained film in vacuum and rehydrating with PBS at 37 °C, the final solution was sonicated for 15 min and filtered through a 0.22 μm sterile poly(ether sulfone) syringe filter to remove crystalline MTO or C6. MTO- or C6-loaded MCT micelles (MTO-MCT and C6-MCT, respectively) were obtained. The targeting micelles were constructed using the exact same method with part of the MCT being replaced by FCT at 9:2 (MCT: FCT) weight ratio. The targeting MTO- or C6-loaded micelles are referred to as MTO-FMCT and C6-FMCT, respectively. Particle Size, Zeta Potential, and Morphology of MTO-MCT and MTO-FMCT. Mean particle size, zeta potential, and size distribution of MTO-MCT and MTOFMCT were assessed by dynamic light scattering (90 Plus Particle Size, Brookhaven Instruments Co. NY, U.S.A.). MTOMCT and MTO-FMCT were stored for 1 month at 4 °C, and their variations in particle size, as well as size distribution, were examined. In addition, micelles’ morphology was inspected using transmission electron microscopy (TEM). Images of phosphotungstic acid (2.0%, w/v) negative-stained cells were taken on a 160 kV JEOL TEM-2010HR (JEOL Ltd., Japan). Drug Encapsulation Efficiency (EE) and Drug Loading (DL). The amount of MTO incorporated into micelles was determined by HPLC. The freeze-drying method was first used to dispose of 1 mL of micelles, and then the powder was dissolved in DCM. After DCM evaporation, the drug was dissolved by adding 3 mL of mobile phase and filtered using a 0.45 μm filter prior to HPLC analysis. A UV−vis detector was used to monitor the column effluent at 662 nm. The physical entrapment efficiency (EE), physical drug loading amount (DLp), chemical drug loading amount (DLc), and total drug loading amount (DLt) were calculated by using the following formula: EE (%) = (amount of MTO physically encapsulated in micelles/initial amount of the free MTO used in the micelles) × 100, DLp (%) = (amount of MTO physically encapsulated in micelles)/(amount of MTO loaded micelles) × 100%, DLc (%) = (amount of MTO chemically conjugated in micelles)/ (amount of MTO loaded micelles) × 100%, DLt (%) = (amount of MTO physically encapsulated in micelles + amount of MTO chemically conjugated in micelles)/(amount of MTO loaded micelles) × 100%. pH-Sensitivity of MCT. pH-sensitivity of MCT was determined as reported elsewhere.21 Briefly, MCT with MTO concentration equivalent to 200 μg/mL, was sealed into a dialysis bag with MW 1000 Da and subsequently dialyzed against 40 mL of phosphate buffer (pH 3.0, 5.0 and 7.4). At defined time points, samples were collected, and equivalent amounts of blank media were added. The amount of released drug was assessed using HPLC. In Vitro Controlled Drug Release. In vitro drug release of MTO-MCT and MTO-FMCT micelles was performed in a 0.1% w/v Tween-80-complemented PBS buffer (pH 5 and 7.4). Briefly, 4 mL of MTO-MCT or MTO-FMCT was placed into a dialysis bag with a MW cutoff of 1000 Da, put into 45 mL of PBS buffer at 37 °C, and stirred at 100 rpm. Samples were taken out and replaced with equals amounts of fresh medium at different time intervals. The withdrawn media comprising the

laminopyridine (DMAP), and N-hydroxysuccinimide (NHS) were all obtained from Sigma-Aldrich Shanghai Trading Co, Ltd., China. Coumarin-6, dimethyl sulfoxide (DMSO), trypsinEDTA solution, triethylamine (TEA), MTT, phosphatebuffered saline (PBS, pH 7.4), RPMI 1640 medium, penicillin−streptomycin solution, DAPI, and fetal bovine serum (FBS) were all purchased from Sunshine Biotechnology Co., Ltd. (Nanjing, China). Tween-80 was provided by Sinopharm Chemical Reagent Co., Ltd. All other solvents were of HPLC grade. MCF-7 human breast cancer cell lines were obtained from American Type Culture Collection (ATCC). 1,1′-Dioctadecyltetramethyl indotricarbocyanine iodide (DiR) was obtained from Sigma-Aldrich Shanghai. Cell Culture. MCF-7 human breast cells and hCMEC/D3 BBB cells were used as models. The cells lines were nurtured in a RPMI 1640 medium under 37 °C in a 5.0% CO2 humidified atmosphere. The medium, which contained 1% penicillin− streptomycin solution and 10% FBS, was renewed every second day until 80% confluence was obtained. Before the experiment, once the precultured cells had reached 75% confluence, the cells were rinsed with PBS and trypsinized before harvest. Animals. The male Sprague−Dawley rats weighing about 220 g and the female athymic nude mice were obtained from the Shanghai Silaike Laboratory Animal Limited Liability Company. All the animals were subjected to regular 12 h light−dark cycle and kept in a controlled temperature and humidity standard animal room. Approval for the methods used was given by the Animal Ethics Committee of China Pharmaceutical University. Synthesis and Characterization of TPGS2k, FCT, and MCT. Appropriate volume of dichloromethane (DCM) was used to dissolve D-α-TOS and bis-amine PEG2k along with EDC, NHS, and DMAP at a stoichiometric ratio of 1:1.2:1.5:1.5:0.1, respectively. The mixture was supplemented with 20 μL of TEA and gently stirred for 2 days under a nitrogen stream. After eliminating the byproducts through filtration, the mixture was subjected to precipitation in diethyl ether. Subsequently, the precipitate was dissolved in water and subjected to evaporation to remove residual organic solvent then dialyzed against water. The final product, D-α-tocopheryl amino polyethylene glycol 2k succinate (TPGS2k-NH2), was collected after lyophilization.9 The aforesaid procedure was used to synthesize FCT. Accurate amounts of TPGS2k-NH2, FA, EDC, and NHS at a stoichiometric ratio of 1:1:1.5:1.5, respectively were mixed. Freeze-drying of the final product was carried out after the mixture was purified.9,15 MCT was synthesized using the same above-mentioned method. First, MTO and succinic anhydride (SA) were reacted to obtain MTO disuccinic ester (MTO-SA) with a yield of 83%. Under nitrogen protection and room-temperature conditions for 24 h, MTO-SA was reacted with TPGS2k-NH2, EDC, and NHS at a stoichiometric ratio of 1:1:1.5:1.5, respectively, in DMSO. In order to remove unreacted MTO and surplus of reagents, the solution was sealed into a sterile MWCO 1000 Da dialysis bag, put into DMSO for 24 h, and then further dialyzed with water for another 48 h to completely eliminate DMSO. The final mixture was cryodesiccated to obtain a blue powder. The conjugation ratio of TPGS2k-NH2 with MTO-SA was assessed using 1H NMR, whereas the purity of MCT was determined by HPLC at 242 nm. All synthesis steps are depicted in Figure S1. Chemical conjugation was confirmed by 1H NMR and FTIR studies. 1084

DOI: 10.1021/acs.molpharmaceut.6b01009 Mol. Pharmaceutics 2017, 14, 1082−1094

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Molecular Pharmaceutics

1440 min after IV administration. All samples were collected in heparinized tubes and subjected to centrifugation at 6000 rpm for 10 min; samples of plasma were drawn and stored at −20 °C for subsequent analysis. In Vivo Distribution Behavior of MCT and FMCT Micelles for Tumor-Targeting Studies. Tumor targeting was explored in nude mice bearing subcutaneous tumors of MCF-7 cells. To construct the tumor xenograft model, MCF-7 cells (5 × 106 cells/mL) suspended in saline medium were inoculated subcutaneously in the left armpit site of nude mice. Tumor volume (V) was evaluated by measuring width (W) and length (L), then determined as V = L × W2/2. The tumorbearing mice whose tumors have reached a volume of about 200 mm3 were subjected to this experiment. DiR, as the nearinfrared fluorescent probe, was used to load in MCT and FMCT micelles (DiR-MCT and DiR-FMCT) with the same process as described above with MTO replaced with DiR. Fluorescent micelles were intravenously injected at a dose corresponding to 2 mg/kg of DiR. After the animals were anesthetized, in vivo imaging tests were performed by collecting pictures within the fixed excitation wavelength of 720 nm and emission wavelength of 790 nm. The exposure time was 20 s. Planar pictures of fluorescence were taken at different points (0.5, 1, 2, 4, 6, 8, 12, and 24 h). Finally, 24 h after intravenous administration, mice in each group were euthanized by cervical dislocation, and heart, liver, lung, spleen, kidney, brain, and tumor tissues were removed, washed with saline, and dried using filter paper. The same in vivo imaging photo collection conditions were applied to evaluate the distribution behavior of DiR-MCT and DiR-FMCT in tissue level. The mean fluorescence intensity was normalized to the weight of the tissue. Moreover, tumor tissues were sectioned with a freezing microtome and observed with CLSM after staining with DAPI. In Vivo Antitumor Activity. Once the tumor volume was 100 mm3, MCF-7 tumor-bearing mice were chosen and used as experimental models. The nude mice bearing MCF-7 tumor cells were randomly separated into four groups (n = 10), where three groups were administered MTO solution, MTO-MCT, and MTO-FMCT at a dose corresponding to 2 mg/kg of MTO. The first administered dose was designated as day 0, and subsequent doses were given on days 2, 4, and 6, respectively. The fourth group received saline as a control group. The antitumor activity was assessed by regularly monitoring the tumor volume following the first administration. Body weight and survival rate were also assessed. In order to determine the apoptotic effect of MTO-MCT and MTO-FMCT on nude mice bearing a tumor xenograft, mice were first sacrificed by cervical dislocation after 14 days of observation. Removal of the tumors was then performed. After the tumor tissues were fixed and frozen, the resulting sliced samples were processed by the TUNEL staining method to detect apoptotic cells and calculate the percentage of apoptotic cells. Additionally, heart, lung, liver, kidney, and spleen were withdrawn, transformed to paraffin sections, processed using hematoxylin-eosin (H&E) stain in order to observe the pathological changes of the above-mentioned tissues.

released drug were analyzed by the same above-mentioned HPLC procedure (n = 3). In Vitro Concentration-Dependent Uptake and TimeDependent Uptake. After reaching about 80% confluence as a minimum, MCF-7 tumor cells were inoculated into 24-well plates at a density of 105 cell/well and cultivated using a 10% FBS-supplemented RPMI 1640 medium. The culture media were replaced by suspensions of C6-MCT or C6-FMCT at a C6 concentration of 25, 50, 100, and 200 ng/mL (n = 3). After the plates were incubated at 37 °C for 4 h, C6-MCT or C6FMCT suspensions were first aspirated from the wells, and then PBS was used three times to eliminate the untrapped micelles. At preselected intervals, cells were rinsed, harvested using 150 μL of trypsinized RPMI 1640 medium (50%), and their uptake was quantitatively assessed by flow cytometry. In order to evaluate the effect time might have on MCF-7 micelles uptake, wells were supplemented with 200 μL of C6-MCT or C6-FMCT at a C6 concentration of 100 ng/mL. After adjusting the volume to 400 μL with RPMI 1640 FBS-free medium, the plates were incubated for 0.5, 1, 2, and 4 h at 37 °C. After incubation, the same process as described above was applied, and cells were analyzed with a flow cytometer. Different concentrations of FA (0, 1, 10, and 100 μM) were used to inhibit FA-receptor-mediated endocytosis. After incubation, the same above-mentioned analysis procedure was carried out. Confocal Laser Scanning Microscopy Analysis. Briefly, MCF-7 cancer cells were first dispensed at a density of 105/ dish. After the cells were nurtured for 24 h, FBS-free media dissolving C6-MCT and C6-FMCT at C6 concentration of 100 ng/mL were added to substitute the culture media and then incubated for 1 and 4 h. In order to investigate the competitive inhibition of C6-FMCT uptake, cells were incubated for 4 h in C6-FMCT-containing medium after an initial 1 h treatment with free FA (1 μM). Cells were then rinsed with PBS, fixed with 4% paraformaldehyde, and stained with Hoechst 33342 DNA dye.24 Lastly, a ZEISS LSM 700 SYSTEM confocal laser scanning microscope (ZEISS, Germany) was used to examine the intracellular distribution of micelles. In Vitro Cytotoxicity. Citotoxicity of TPGK2k, MCT, FCT, free MTO, MTO-MCT, or MTO-FMCT on MCF-7 cells, and hCMEC/D3 BBB cells was investigated using the MTT method. First, after the cells were dispensed into 96-well plates at a density of 5 × 103 cells/well and incubated at 37 °C for 24 h, culture medium was substituted with different concentrations of serum-free culture media dissolving TPGK2k, MCT, FCT, free MTO, MTO-MCT, or MTO-FMCT. After incubation and then addition of 10 μL of MTT solution (5 mg/ mL), cells were further incubated for 4 h. The supernatant was removed, and blue formazan crystals were dissolved in DMSO. The absorbance in each well was measured at 570 nm. The viability percentage was assessed using the following formula: cell viability (%) = [(A − An)/(Ap − An)] × 100%; where As was absorbance value of each sample well, An was the absorbance of negative-control wells (PBS), and Ap was the absorbance value of positive-control wells (medium). In Vivo Pharmacokinetics. To explore the pharmacokinetics of MTO injection, MTO-MCT and MTO-FMCT in vivo, male Sprague−Dawley rats weighing about 220 g were randomly separated into three groups (n = 6) and MTO injection, MTO-MCT or MTO-FMCT were administered intravenously at an MTO dose equivalent to 2 mg/kg. Blood collection was performed by retro-orbital puncture with the aid of a glass capillary at 5, 15, 30, 60, 120, 240, 360, 480, 720, and

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RESULTS AND DISCUSSION Characterization of TPGS, MCT, and FCT Conjugates. Synthesis of MCT and FCT conjugates was confirmed by 1H NMR (Figure S2) and FTIR spectroscopy (Figure S3). In the TPGS2k 1H NMR spectrum, the characteristic peak at 3.60 ppm could be associated with the methylene hydrogen atoms 1085

DOI: 10.1021/acs.molpharmaceut.6b01009 Mol. Pharmaceutics 2017, 14, 1082−1094

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Molecular Pharmaceutics Table 1. Physicochemical Properties of MTO-MCT, MTO-FMCT, and MTO-TPGS2k (n = 3) formulations

particle size (nm)

PIa

zeta potential(mV)

EE (%)b

DLp (%)c

DLc (%)d

DLt (%)e

MTO-MCT MTO-FMCT MTO-TPGS2k

78.13 ± 1.96 90.76 ± 3.21 93.02 ± 5.17

0.19 ± 0.05 0.26 ± 0.04 0.186 ± 0.08

−9.62 ± 0.05 −17.72 ± 1.41 −4.52 ± 2.15

73.17 ± 2.63* 61.50 ± 4.20* 9.98 ± 4.32

9.51 ± 0.60* 6.53 ± 2.35* 0.60 ± 1.46

7.40 ± 0.05 6.26 ± 0.16 0

16.92 ± 0.55* 12.78 ± 2.19* 0.60 ± 1.46

a

PI: Polydispersity index. bEE: Physical entrapment efficiency. cDLp: Physical drug loading amount dDLc: Chemical drug loading amount eDLt: Total drug loading amount *vs MTO-TPGS2k, p < 0.05.

Figure 1. Particle size and TEM images of MTO-TPGS2k, MTO-MCT, and MTO-FMCT.

(O−CH2−CH2−) of PEG block. Moreover, after comparing the TPGS spectrum with that of α-TOS, we could clearly see that the characteristic COOH peak (12.3 ppm) of α-TOS disappeared, which indicated that the carboxylic group reacted with the amine group of PEG-(NH2)2 through a carbonimide reaction, leading to the formation of an amide group. Also, the characteristic triple methylbenzene peaks of α-TOS could still be seen on the TPGS spectrum at 1.9−2.0 ppm, which confirmed the success of the synthesis. Furthermore, we noticed the appearance of a broad peak at around 6 ppm, corresponding to the amide function’s hydrogen. For MTO-SA, the peaks at 13.43 and 10.58 ppm seen in the 1 H NMR spectrum were considered as the characteristic peaks of aryl−OH and aryl-NH, respectively. After conjugation with TPGS2k, the characteristic peaks at 13.60, 10.66, and 3.51 ppm were observed, showing that MTO-SA was successfully conjugated to TPGS2k and formed MCT (Figure S2). Additionally, the integrated peak at 13.60 ppm (−OH) corresponds to 1H, whereas the integrated area of the triplet peak at 1.88−2.00 ppm (methylbenzene of vitamin E) corresponds to 9H. Because MTO contains two aryl−OH groups, peak represents in reality two hydrogen protons, which makes the peak “d” represent 18 hydrogen protons. This suggested that MTO-SA was linked to TPGS2k entities in a ratio of 1:2 (mol/mol). The purity of MCT, determined by HPLC, was about 98% (Figure S4). The amount of MTO chemically conjugated to MCT could be calculated from the amount of MCT used. FCT 1H NMR spectrum clearly exhibited signals from both TPGS and FA showing a typical peak at 3.5 ppm, that is, distinctive of −OCH2CH2− (methylene protons) of PEG block

in TPGS. Peaks between 6.6 and 8.7 ppm belonged to the aromatic CH in FA.25 However, the hydrogen peak of the carboxyl group in FA that was observed at 12.28 ppm in FA spectrum had completely disappeared in FCT spectrum, which revealed that the carboxylic group was involved in a reaction and resulted in the formation of an amide function. Moreover, the shift of the peak from 8.13 to 7.91 ppm and the peak at 11.4 ppm being replaced by a smaller peak also provided another proof of FA-TPGS2k conjugation. Similarly, the FTIR spectra confirmed the results obtained from the 1H NMR spectra. As shown in the TPGS2k FTIR spectrum (Figure S3), the amide function formation between D-α-TOS and PEG (NH2)2 was proven by the partial overlapping of CO stretch at 1655 cm−1 on the C−N stretch at 1554 cm−1. On the other hand, the MTO-SA FTIR spectrum showed that the peak at 3318.6 cm−1, corresponding to the OH functional group of MTO, was reduced and shifted to 3446.3 cm−1. Also, the peak appearing at 1730 cm−1 (i.e., COOR) in the MTO-SA spectrum certified the esterification of MTO with succinic anhydride. Finally, the band appearing at 1625−1638 cm−1 in MCT spectrum was characteristic of the amide function (CO), thus confirming the conjugation of MTO-SA to TPGS2k. For FCT, the presence of peaks at 1470 and 1605 cm−1 represented the absorption of the phenyl ring of FA and the N−H bending, respectively. Whereas the aromatic ring-stretching and N−H bending (−CONH) of FA can be observed at 2958 and 1514 cm−1, respectively. The CMC of TPGS2k, MCT, and FCT were found to be 0.0251, 0.072, and 0.0338 mg/mL, respectively, which were substantially lower than that of TPGS1k (0.2 mg/mL). Owing to the lower CMC, TPGS2k micelles were expected to confer 1086

DOI: 10.1021/acs.molpharmaceut.6b01009 Mol. Pharmaceutics 2017, 14, 1082−1094

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Molecular Pharmaceutics

Figure 2. (A) MTO dissociation from MCT conjugate incubated in phosphate buffer with different pH values at 37 °C (mean ± SD and n = 3). (B) Quantitative analysis of the in vitro release of MTO from the MTO-MCT and MTO-FMCT incubated in phosphate buffer pH 5 and 7.4 at 37 °C (mean ± SD and n = 3).

Figure 3. (A,B) Comparison of C6-MCT and C6-FMCT accumulation into MCF-7 tumor cells after incubation at different concentrations and for different periods of time. Data represent mean ± SD (n = 6). (C) Uptake of C6-MCT and C6-FMCT micelles in the presence of different FA concentrations to saturate the FA receptor and thus inhibiting the receptor-mediated endocytosis. Data represent mean ± SD (n = 6).

As shown in Table S1, stability studies of MTO-MCT and MTO-FMCT showed insignificant changes in the particle size or size distribution within 6 months, suggesting that the MTOloaded micelles remained relatively stable during this period. In Vitro Controlled Drug Release. Before investigating the in vitro release of MTO from micelles, we first evaluated MTO release from MCT at different pH levels. As demonstrated in Figure 2A, MCT did not exhibit any drastic initial burst release, which demonstrates the strength of the amide bond. Longer incubation times showed that the drug release from MCT was faster as the pH values was decreasing. After 24 h, at pH 3.0 MTO released rapidly from the conjugate, reaching 48.5 ± 3.2%, whereas its release was considerably slower at pH 5.0, reaching only 36.1 ± 2.3% (p < 0.01). Meanwhile, MTO release at pH 7.0 was, as expected, barely 9.6 ± 0.2%. These results show that our micellar formulations could pH-responsively control the drug release of MTO. The conjugation of polymers to drugs to control drug release, in most cases by simple hydrolysis, is an important feature that has already been reported several times.21,29,30 The in vitro drug release obtained revealed that the MCT conjugate was sufficiently stable during circulation (pH 7.4) to deliver MTO to cancer cells and labile under mild acidic conditions, releasing the drug at the tumor site. In vitro release of MTO from micelles is shown in Figure 2B. The initial drug release at pH 5.0 was 35.2% from MTO-MCT and 40.13% from MTO-FMCT in the first 12 h. However, at pH 7.4, it was only 16.97% from MTO-MCT and 19.78% from MTO-FMCT. This rapid release in comparison to that of MCT could be attributed to the partial release of the physically loaded MTO from the micelles. After 40 h, the cumulative percentage

greater stability for MTO and enhanced resistance to dissociation in bloodstream.9 Physicochemical Properties of Micelles. Particle size and zeta potential of the prepared micelles were obtained using DLS and are shown in Table 1. The mean sizes of MTO-MCT and MTO-FMCT were 78.13 ± 1.96 and 90.760 ± 3.21 nm, with a zeta potential of −9.62 ± 0.051 and −17.72 ± 1.41 mV, respectively. The increase in the negativity of zeta potential of MTO-FMCT might be due to the presence of carboxyl group in FA. The TEM images of MTO-MCT and MTO-FMCT revealed spherical shapes with homogeneous particle sizes, which conforms to the previously obtained results by DLS (Figure 1). MTO-MCT and MTO-FMCT sizes are found to be in the suitable size range that would allow a much-increased tumor tissue accumulation through enhanced permeability and retention (EPR) effects.26−28 Drug encapsulation efficiency, which is of high relevance with respect to drug delivery systems, was 73.17 ± 2.63% and 61.5 ± 4.2% for MTO-MCT and MTO-FMCT, respectively. However, the physical drug loading ratios of MTO-MCT and MTOFMCT were 9.513 ± 0.60 and 6.534 ± 2.35%, respectively. That represented the corresponding 15.85- and 10.88-fold increase compared with that of MCT (Table 1). High drug loading was attained because of the similarity of the chemical structures as MCT and FMCT had MTO in their compositions (“like dissolves like” rule).23 Notably, MTO-MCT displayed a higher DLc than that of MTO-FMCT. This could be explained by the fact that MTO-FMCT has a lower ratio of MCT compared with MTO-MCT. This results in a lower DLc and consequently in a lower DLp capacity, which is probably due to the above-mentioned “like dissolves like” rule. 1087

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Figure 4. Confocal laser scanning microscopy of MCF-7 cells incubated with C6-MCT and C6-FMCT for 1 and 4 h and C6-FMCT in the presence of folic acid as inhibitor for 4 h.

drug release at pH 5.0 was 76.96% and 86.96% for MTO-MCT and MTO-FMCT, respectively, and was 36.26% for MTOMCT and 42.26% for MTO-FMCT at pH 7.4. Both of the formulated micelles exhibited a pH-sensitive release pattern; targeted micelles showed an insignificant slightly higher drug release. We expect that the fast-physical release would be a practical tool to hinder the tumor cells growth at the commencement of treatment. Furthermore, the cumulative release due to the continuous physical release and slow chemical release, would confer a sustainable treatment of tumor cells.9 At pH 7.4, MTO-MCT and MTO-FMCT formulations had good stability and slow drug release, which is promising for minimizing drug release in the general circulation and thus limiting its undesirable side effects. At pH 5.0, the patterns showed a practically complete drug release from the micelles over 40 h. This accelerated drug release might also occur inside the endo/lysosomes of cancer cells ( 0.05). At the concentration of 5 μg/mL, MCT, MTO-MCT, FMCT, and

Figure 6. Mean plasma concentration of MTO versus time curves after tail vein injection in SD rats of MTO solution, MTO-MCT, and MTO-FMCT. Each data represents Mean ± SD measured at each time point (n = 5). 1089

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Molecular Pharmaceutics Table 2. Mean Values of Pharmacokinetic Parameters of MTO (n = 5)a

a

preparation

MTO

MTO-MCT

MTO-FMCT

AUC (ng/mL·h) t1/2 (h) MRT (h)

306.282 ± 102** 0.31 ± 0.15** 0.42 ± 0.23**

4026.783 ± 1908* 12.57 ± 2.26* 16.14 ± 3.68*

4926.526 ± 1895 13.65 ± 2.29 19.08 ± 2.73

Data are represented as mean ± SD (n = 5).

**

P < 0.05, MTO vs MTO-MCT or MTO-FMCT. *P > 0.05, MTO-MCT vs MTO-FMCT.

Figure 7. (A) Featured in vivo real-time imaging fluorescence images of MCF-7 tumor-xenografted nude mice observed at 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h postinjection of DiR-MCT and DiR-FMCT micelles at DiR doses of 0.5 mg/kg. Arrows indicate the tumor site. (B) Ex vivo tumor targeting of DiR-MCT and DiR-FMCT in MCF-7 xenograft tumor model; fluorescence images of major organs isolated from nude mice bearing MCF-7 xenograft tumor 24 h after intravenous injection. (C) Semi quantification of DiR-MCT and DiR-FMCT in the separated organs. All data are represented as mean ± SD (n = 3). *P < 0.05, DiR-MCT vs DiR-FMCT.

more advantageous in inhibiting the proliferation of tumor cells. In addition, the anticancer drug MTO uptake by a noncarrier-mediated passive diffusion explains its toxicity on both tested cell lines.43,44 MTO directly interacts with the cytoplasmic enzymes and proteins that were reported responsible for its side effects,20,45,46 for instance, mitochondrial toxicity.47,48 We expect that these side effects could be taken under control after conjugation. In Vivo Pharmacokinetics. MTO plasma concentration− time profiles were obtained after intravenous administration of free MTO, MTO-MCT, and MTO-FMCT (equivalent dose of 2 mg/kg MTO) in male SD rats. Figure 6 indicates that MTO plasma concentration declined rapidly following the intravenous administration of free MTO and was nearly undetectable 1 h later. However, after the MTO was conjugated to TPGS2k and also loaded into the core of the micelles, the plasma clearance rate decreased greatly, indicating that MTOMCT and MTO-FMCT played a rather protective effect for

with TPGS2k through MTO’s double hydroxyl groups, the cytotoxicity of MTO would be concealed and would be only restored after cellular internalization, hydrolysis, and release of the drug from the conjugate. Thus, the different effects between hCMEC/D3 cells and MCF-7 cells could be attributed to the following reasons. First, the lack of FR expression in hCMEC/ D3 cells41 compared to MCF-7 cells led to a low uptake of FMCT micelles. Second, the increased cytotoxicity of MCT and FMCT toward MCF-7 cells compared to hCMEC/D3 cells could be associated with the fact that they are more internalized by MCF-7 because of the high proliferation ability, higher requirement for nutrients, and the relatively strong metabolic capability of MCF-7 cells. These properties also allow the micelles to effectively release MTO to exert its toxic effect. Furthermore, in comparison with normal cells, the recycling route in tumor cells is basically absent, consequently inducing an endo/lysosomes accumulation in tumor cells42 and thus suggesting the pH-sensitive degradation of MCT would be 1090

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Figure 8. CLSM imaging for tumor tissue section 24 h after intravenous injection.

where a clearer DiR signal was distributed within the nucleus of the mice treated with DiR-FMCT. The results were in accordance with our cellular uptake study. Because of their enhanced cellular uptake, pharmacokinetic properties, and tumor-targeting delivery abilities, MTO-loaded micelles showed a greater inhibitory effect on the tumor growth compared to the free MTO injection (p < 0.05) (Figure 9A). Moreover, all MTO formulations exhibited a shrinking effect on tumor volume with different rates (MTO-FMCT > MTOMCT > MTO solution). As tissue distribution studies showed (Figure 8A), FMCT micelles significantly increased the retention and accumulation into tumor sites, which resulted in a stronger pharmacodynamics effect. Furthermore, because MTO solution has a strong toxicity on the heart and the liver, the continuous administration led to a sharp weight loss of the mice group that received the free MTO injection, which also exhibited the shortest survival time (Figure 9B). Despite the fact that MTO-MCT and MTOFMCT also induced a slight weight loss (Figure S5), however, MTO-MCT and MTO-FMCT significantly prolonged the nude mice survival time and offered better efficacy/toxicity ratios. The apoptosis analysis of tumor tissues showed that most of the tumor cells in the saline group were normal, whereas the amount of cell apoptosis induced by continuous drug administration was in the following order: MTO-FMCT > MTO-MCT > MTO. These results were consistent with those of the tumor growth inhibition. In addition, it can be seen from TUNEL images (Figure 9C) that most cells (>90%) in the

MTO. The pharmacokinetic parameters in Table 2 show that compared to free MTO, injection both MTO-MCT and MTOFMCT exhibited prolonged half-lives (T1/2) of 11.5 and 13 times higher, respectively (p < 0.05), and mean residence times (MRT) 14.1 and 13.3 times higher, respectively (p < 0.05). The extended circulation time via TPGS conjugation could improve the EPR effect, and hence resulting in an enhanced therapeutic capacity.49 The area-under-the-curve (AUC), which is a major indicator of a drug therapeutic efficacy, was improved by 9.7 times for MTO-MCT and by 5.8 times for MTO-FMCT (p < 0.05). Moreover, MTO-MCT and MTO-FMCT, based on their similar pharmacokinetic behaviors, could reduce the administered doses and at the same time could prevent MTO from being metabolized by protecting its hydroxyl groups,47 thus prolonging its plasmatic half-life time and reducing its undesirable toxic effect related to its metabolites.48 In Vivo Biodistribution and Pharmacodynamics Evaluation. In vivo imaging study (Figure 7A) showed that DiR-MCT and DiR-FMCT exhibited a strong fluorescence signal at the MCF-7 tumor sites, where the strongest signal was between 4 to 6 h. As time went by, signals gradually weakened. Because of the FA receptor-mediated targeting of tumor cells, DiR-FMCT could further penetrate the tumor cells and showed a higher tumor accumulation capacity compared to DiR-MCT. Ex vivo fluorescence images of tumors and major organs collected 24 h postinjection are shown in Figure 7B. The DiRFMCT fluorescence signal in tumor tissue was stronger than that of DiR-MCT. Moreover, from the tissue sections of the tumor (Figure 8), a similar phenomenon could also be noticed, 1091

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Figure 9. (A) Tumor growth curves of MCF-7 tumor-bearing nude mice following administration of various MTO preparations at a 2 mg/kg dose. (B) Survival rates of nude mice bearing MCF-7 cells 30 days postinjection of different MTO formulations at a 2 mg/kg dose. (C,D) Representative images of MCF-7 Cells apoptosis in tumor tissues induced by different MTO formulations at a 2 mg/kg dose studied by TUNEL assay and H&E stain. *P < 0.05, Saline vs MTO injection, MTO-MCT or MTO-FMCT; #P < 0.05, MTO injection vs MTO-MCT or MTO-FMCT.

toxicity while being a vehicle for targeted delivery of MTO. The physicochemical properties and drug release of MTO-loaded micelles (MTO-MCT and MTO-FMCT, respectively) were characterized and showed excellent drug loading capacity, enhanced stability, and satisfactory pH-sensitive drug release. Cellular uptake and antitumor efficacy of MTO-MCT and MTO-FMCT in vitro revealed that MTO-FMCT exhibited improved uptake via folate-mediated endocytosis, leading to a higher cytotoxic effect toward MCF-7 cells. More importantly, the enhanced pharmacokinetic behavior and receptor-mediated tumor targeting potential of the MTO-FMCT remarkably increased their accumulation in tumors and thus induced more tumor cell apoptosis and exhibited a higher in vivo antitumor potency compared with MTO-MCT and free MTO. MTOFMCT also had a lower toxicity toward major organs. The present findings demonstrated that MTO-FMCT may be used as a potential drug delivery system for targeted breast cancer therapy.

MTO-FMCT group were apoptotic and necrotic, and the apoptosis ratio was significantly higher than that of MTOMCT. Results available from H&E stain (Figure 9D) were similar to those obtained in apoptosis analysis. The tumor tissue stains of MTO-FMCT group exhibited only a small fraction of surviving cells, whereas the abundant acidophilic cytoplasm indicated a high cell death ratio. For MTO-FMCT, both passive- and active-targeting mechanisms expanded MTO’s in situ drug distribution and accumulation in the tumor, which effectively inhibited and reduced the tumors’ growth. Safety evaluation by H&E analysis in major organs and tissues showed that MTO injection had a certain influence on the cardiomyocytes, liver, and spleen (Figure S6), whereas slight toxicity in these tissues was observed in MTO-MCT- or MTO-FMCT-treated groups. Furthermore, compared with the saline group, MTO, MTO-MCT, and MTO-FMCT had no apparent pathological toxicity on the lungs and kidneys. These findings support that MTO loading into micelles would promote a better efficacy/toxicity ratio.

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ASSOCIATED CONTENT

S Supporting Information *

CONCLUSIONS In this study, MTO- or FA-conjugated TPGS2k micellar materials (MCT and FCT, respectively) were successfully designed and synthesized to specifically diminish systemic

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b01009. 1092

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Synthesis schema of MCT and FCT; 1H NMR spectra of TPGS, MTO-SA, MCT, and FCT; FTIR spectra of TPGS, MTO-SA, MCT, and FCT; HPLC of MTO and MCT detected with a UV−vis detector; determination of the critical micelle concentration (CMC); storage stability of MTO-MCT and MTO-FMCT micelles; storage stability of lyophilized MCT and FCT micelles; body weight curve of MCF-7 tumor-bearing nude mice; representative images of H&E stain of different tissues from MCF-7 xenograft tumor nude mice (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail for Z.S.: [email protected]. Phone: +86 25 83271076. Fax: +86 25 83271076. *E-mail for Q.P.: [email protected]. Phone: +86 25 83271092. Fax: +86 25 83271092. ORCID

Nida El Islem Guissi: 0000-0003-4425-7580 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Funds for NSFC Program No. 81273467. REFERENCES

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