Self-Assembled Nanoparticles of Amphiphilic Twin Drug from

May 21, 2015 - We report here an amphiphilic twin drug strategy directly using small molecular hydrophilic and hydrophobic anticancer drugs to self-as...
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Self-Assembled Nanoparticles of Amphiphilic Twin Drug from Floxuridine and Bendamustine for Cancer Therapy Ting Zhang, Ping Huang, Leilei Shi, Yue Su, Linzhu Zhou, Xinyuan Zhu,* and Deyue Yan* School of Chemistry and Chemical Engineering, Shanghai Key Lab of Electrical Insulation and Thermal Aging, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China S Supporting Information *

ABSTRACT: We report here an amphiphilic twin drug strategy directly using small molecular hydrophilic and hydrophobic anticancer drugs to selfassemble into nanoparticles with a high and fixed drug content, which can solve problems of anticancer drug delivery including poor water solubility, low therapeutic indices, and severe side effects. The twin drug has been prepared by the esterification of the hydrophilic anticancer drug floxuridine (FdU) with the hydrophobic anticancer drug bendamustine (BdM). Due to its inherent amphiphilicity, the FdU−BdM twin drug can self-assemble into stable and well-defined nanoparticles. After FdU−BdM twin drug enters into cells, the ester linkage between hydrophilic and hydrophobic drugs is readily cleaved by hydrolysis to release free FdU and BdM. Since both FdU and BdM can kill cancer cells, the FdU−BdM twin drug nanoparticles can overcome the multidrug resistance (MDR) of tumor cells and present an excellent anticancer activity. This strategy can be extended to other hydrophilic and hydrophobic anticancer drugs to synthesize amphiphilic twin drugs which can form nanoparticles to self-deliver drugs for cancer therapy. KEYWORDS: twin drug, floxuridine, bendamustine, nanoparticle, cancer therapy

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surfactants18,19 and polymers,20−22 it can be inferred that the amphiphilic twin drug consisting of hydrophilic FdU and another hydrophobic drug might form nanoparticles by selfassembly to exert advantages from twin drug and nanoparticles. In this work, we synthesized a novel amphiphilic twin drug of FdU and BdM. FdU is a hydrophilic 5-fluorouracil derivative and has a specific activity in DNA and less cytotoxicity in RNA, and BdM is a hydrophobic bifunctional alkylating agent that has potential antimetabolite properties.23−26 We conjugated the hydrophilic FdU and hydrophobic BdM by ester bond to obtain a twin drug. Due to its inherent amphiphilicity, the FdU−BdM twin drug could self-assemble into stable and welldefined nanoparticles in water. After cellular uptake, the free FdU and BdM are released, attributed to the ester bond between hydrophilic and hydrophobic drugs being readily cleaved by hydrolysis in the acid environment of tumor cells. The FdU−BdM twin drug nanoparticles could overcome the MDR of tumor cells and present an excellent anticancer activity, which develops a new method to self-deliver anticancer drugs.27−29

INTRODUCTION Chemotherapeutic drugs are very critical to cancer therapy. Among them, floxuridine (FdU) is a very promising drug with high potency in the treatment of various tumoral diseases for the past 50 years such as colorectal cancer, liver cancer, and colon cancer.1−4 Although FdU is clinically effective, it exhibits various side effects as a result of its low tumor selectivity, adverse effects at nontumor site actions, and low efficiency of cellular uptake.5 Therefore, the strategies which can improve the efficiency of cellular uptake of FdU and reduce its toxicity are very attractive. During the past decades, prodrug strategy has been extensively adopted to improve physicochemical properties of FdU and reduce its toxicity.6−12 Especially, twin drugs,13 which are defined as compounds that combine two different drugs in one molecule for synergistic treatment, can produce more potent pharmacological effects because the twin drugs show two different pharmacological activities after they enter into cancer cells. However, both prodrugs and twin drugs of FdU belong to small molecule anticancer drugs, which still suffer from several limitations of free anticancer drugs including rapid clearance and premature degradation, low accumulation in tumors, and severe multidrug resistance (MDR) due to their small molecular size. We note that nanotechnology has been widely used in cancer therapy to solve these limitations, ascribed to the nanoscale characteristics and enhanced permeability and retention (EPR) effect of self-assembled nanoparticles.14−17 Inspired by assembly from amphiphilic © XXXX American Chemical Society

Received: January 2, 2015 Revised: April 30, 2015 Accepted: May 21, 2015

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DOI: 10.1021/acs.molpharmaceut.5b00005 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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EXPERIMENTAL SECTION Materials. 4-(Dimethylamino)pyridine (DMAP, 99%, J&K), N,N′-dicyclohexylcarbodiimide (DCC, 99%, J&K), FdU (98%, Adams), and esterase (18 U/mg, Sigma) were used directly from purchase. BdM hydrochloride was provided by Xi’an Zijingtang Company. Calcium hydride was used for drying N,N-dimethylformamide (DMF) more than 48 h and distilling before use. Triethylamine (TEA) was heated by refluxing with calcium hydride, and then distilled prior to use. Biochemical regents including phosphate-buffered saline (PBS), fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and methyl tetrazolium (MTT) were provided by Sigma-Aldrich (St. Louis, MO). Apoptosis assay kit was provided by Invitrogen (Alexafluor 488 annexin V/dead cell). BCA and caspase-3 activity were evaluated by protein assay kit from Beyotime Institute of Biotechnology. Different polystyrene plates including 6- and 96-well ones were provided by Chinese Sangon Biotech. Unless mentioned, other solvents, compounds, and reagents came from the domestic suppliers and were used without further purification. Measurements. 1H and 13C nuclear magnetic resonance (NMR) spectra were registered on a Varian MERCURY plus 400 spectrometer with dimethyl sulfoxide-d6 (DMSO-d6) as solvent at 298 K. Liquid chromatography−mass spectrometry (LC−MS) was performed on a Waters ACQUITY ultrahigh pressure liquid chromatograph (UPLC), which consisted of a sample manager, a solvent manager, and an electrospray mass spectrometer (Waters Q-TOF Premier, Milford, MA, USA). With the help of potassium bromide pellets, Fourier transform infrared (FTIR) spectra were measured between 4000 and 400 cm−1 by a spectrophotometer (PerkinElmer Paragon 1000). An ultraviolet−visible (UV−vis) spectrometer (PerkinElmer Lambda 20) was used to estimate the UV−vis absorption spectra in the range of 265−400 nm. A fluorescence spectrometer (PerkinElmer LS 50B) was used to measure the fluorescence from 260 to 650 nm. The excitation wavelength was λex = 360 nm. Transmission electron microscopy (TEM, JEOL JEM-100CX-II) was adopted for the morphology observation. The testing voltage was set at 200 kV. The sample preparation for TEM observation is described as follows: (1) dropping particle solution onto copper grids with carbon film; (2) drying the sample before TEM measurement. For dynamic light scattering (DLS), it was performed at room temperature by a Malvern Zetasizer Nano ZS90 apparatus. Here, a 4.0 mW He−Ne laser was used. All samples were operated at λ = 633 nm and tested at a scattering angle of 90°. Synthesis of FdU−BdM Twin Drug. Both BdM (150 mg, 0.38 mmol) and DCC (95 mg, 0.46 mmol) were dissolved in dried DMF (4 mL), and the mixture was stirred at 0 °C. After 30 min, the mixture was dropwise added into a solution of FdU (280 mg, 1.14 mmol), DMAP (5 mg, 0.038 mmol), TEA (53 μL, 0.38 mmol), and DMF (6 mL) in a 25 mL flask, and stirred for 48 h at room temperature in the dark. Then the reaction mixture was filtered to remove a white solid (dicyclohexylurea), and the filtrate was concentrated under vacuum. The crude product was purified by column chromatography using dichloromethane (CH2Cl2) and dichloromethane/methanol (CH2Cl2:CH3OH, 10:1 v/v) as the eluent. The product was collected and the solvent was removed by rotary evaporation to give a yellowy solid (135 mg, 61%). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.80−11.78 (d, J = 4.8 Hz, 1H), 8.21− 8.19 (d, J = 7.2 H, 1H), 7.71−7.69 (d, J = 9.2 Hz, 1H), 7.12−

7.10 (d, J = 9.2 Hz, 1H), 6.89−6.88 (d, J = 2.4 Hz, 1H), 6.12− 6.10 (t, J = 13.2 Hz, 1H), 4.24−4.21 (m, 1H), 3.88 (s, 3H), 3.80 (m, 8H), 3.76 (m, 1H), 3.58 (m, 2H), 3.15−3.11 (t, J = 15.2 Hz, 2H), 2.41−2.37 (t, J = 14 Hz, 2H), 2.09−2.06 (m, 2H), 2.03−1.95 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.92, 157.32, 154.06, 149.29, 143.64, 141.68−139.37 (d, J = 231 Hz), 128.82, 125.57, 111.20, 111.02, 110.39, 100.96, 85.02, 84.26, 70.45, 64.70, 54.05, 41.83, 33.07, 30.32, 25.70, 22.56. ESI-MS m/z (M + H+): calcd 585.1637, found 585.1649 (M + H+). Formation of FdU−BdM Twin Drug Nanoparticles. At room temperature, FdU−BdM twin drug (5 mg) was dissolved in DMF (2 mL). Subsequently, deionized water (4 mL) was put dropwise into the solution and stirred slightly for 30 min. The dialysis was carried out against deionized water for at least 1 day (MWCO = 1000 g/mol) in order to eliminate any residual solvent. Every 4 h, the water was renewed. After the dialysis process, the solution volume increased to 10 mL and its concentration was 0.5 mg/mL. The turbidity appeared in the aqueous solution after dialysis, which indicated that the FdU− BdM twin drug had formed nanoparticles. Measurements of Critical Aggregation Concentration (CAC). The dye solubilization method was adopted to determine the critical aggregation concentration using 1,6diphenyl-1,3,5-hexatriene (DPH) as a UV probe. Here, 5.0 × 10−6 mol/L concentration was used for DPH, while the concentration of 5.0 × 10−5 to 0.15 mg/mL was used for FdU− BdM twin drug solutions. With the help of a UV−vis spectrometer (PerkinElmer Lambda 20), the absorbance spectra of all solutions were recorded at 313 nm. In Vitro Drug Release Study. The drug release was evaluated under a simulated physiological condition. PBS solutions (pH 7.4) with or without esterase and acetate buffer mediums (pH 5.0) with or without esterase were prepared. A total of 3 mL of FdU−BdM twin drug nanoparticles was transferred into a dialysis bag (MWCO = 1000 g/mol). The dialysis bag was put in the flask, immersed by 60 mL of pH 7.4 phosphate buffer or pH 5.0 acetate buffer solutions containing (or not) esterase (5 mg, 30 U/mL), and stirred slightly at 37 °C in the dark. Then, 2 mL of the external buffer was replaced with 2 mL of fresh medium immediately at predetermined time intervals, keeping the sinking condition. Here, the fluorescence intensity of the external buffer was used to analyze the amount of released BdM (QC-4-CW spectrometer, excitation at 360 nm). The amount of released FdU was investigated with UV− vis measurement using a PerkinElmer Lambda 20 UV−vis spectrometer. Cell Cultures. MCF-7 cancer cells (a cell line of human breast carcinoma), HeLa cancer cells (a cell line of human uterine cervix carcinoma), and MCF-7/ADR cells were cultured in DMEM in a humidified atmosphere containing 5% CO2 at 37 °C. DMEM was supplied with 10% FBS, penicillin (50 units/mL), and streptomycin (50 units/mL). In Vitro Degradation Experiment of FdU−BdM Twin Drug Nanoparticles. The 6-well plates were used for the seeding of HeLa cells, reaching a cell density of 5.0 × 105 for every well in 1.5 mL of complete DMEM. After that, the cells were cultured for 1 day, and then treated with FdU−BdM twin drug nanoparticles at a concentration of 40 μM for 6 h at 37 °C. Then, the cell growth medium was removed, and cells were rinsed with cold phosphate-buffered saline (PBS) three times. After that, cells were treated with trypsin in order to collect cells into 15 mL polypropylene centrifuge tubes. The cell B

DOI: 10.1021/acs.molpharmaceut.5b00005 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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incubating the cells for 4 h, the medium containing unreacted MTT was removed carefully. Then, 200 μL of DMSO was added into each well to dissolve the obtained blue formazan crystals, and the absorbance at 490 nm was recorded on a BioTek Synergy H4 hybrid reader to calculate the cell viability. Apoptosis with Flow Cytometry Assays. HeLa cells were seeded in 6-well plates at a density of 5.0 × 105 cells per well in 1.5 mL of complete DMEM and incubated for 24 h. The cells were treated with FdU, BdM, the FdU/BdM mixture, and the FdU−BdM twin drug nanoparticles using the same concentration of 20 μM at 37 °C for 24 h. HeLa cells without the treatment were used as a control. For quantitative measurement of apoptosis, both floating and attached cells were collected and washed three times with cold PBS, stained with Alexa Fluor 488 annexin V and PI according to the manufacturer’s instructions to determine cell apoptosis. The measurements of a BD LSRFortessa flow cytometer were performed to record and analyze the data for 1.0 × 104 gated events. Activity Assay of Caspase-3 Protein. HeLa cells, MCF-7 cells, and MCF-7/ADR cells were seeded in plates at 3.0 × 106 in 10 mL of complete DMEM and cultured for 24 h. The HeLa cells and MCF-7 cells were treated with FdU, BdM, FdU/BdM mixture, and FdU−BdM twin drug nanoparticles (20 μM). For MCF-7/ADR cells, the concentration was 40 μM. Cells without the treatment were used as control. After 24 h incubation, the culture medium was removed, and cells were rinsed by PBS twice. Then, cold PBS (2 mL) was added into each plate, and the cells were scraped from the plate in order to collect cells into a centrifuge tube. The cell suspensions were centrifuged twice at 800 rpm for 8 min at 25 °C. Subsequently, the supernatants were removed. The cells were resuspended in 200 μL of cell lysis buffer and kept on ice for 30 min. At 4 °C, microcentrifuge tubes were adopted for the centrifugation of lysates at 10,000 rpm for 100 s. BCA protein assay was provided to test the content of protein in the supernatant, and then the protein content of every sample was adjusted to the same concentration. 70 μL of the protein solution was distributed to each well of a 96-well plate. Then, 50 μL of 2× reaction buffer (containing 10 mM DTT) and 5 μL of the DEVD-p NA caspase-3 substrate were added into every sample in order to make the final concentration become 200 μM. Then, the protein activity was analyzed by monitoring the absorbance at 405 nm with a hybrid reader (BioTek Synergy H4) after 2 h of incubation at 37 °C. The caspase-3 protein activity was calculated as fold of the measured optical density (OD) values obtained from untreated control cells, and all the values of samples should subtract the OD value of the blank.

suspensions were centrifuged twice at 800 rpm for 8 min at room temperature. Following removal of the supernatants, the cells were resuspended in 2 mL of methanol. Then, the cell suspension was sonicated with alternative cycles of 10 s pulses after every 5 s intervals for 10 min using an ultrasonicator probe (Vibra cell 750). After sonication, the cell suspensions were centrifuged at 4 °C and 1500 rpm for 8 min twice and the supernatant was collected. Finally, the supernatant was analyzed using LC−MS. Cellular Uptake of FdU−BdM Twin Drug Nanoparticles in HeLa Cells. In vitro uptake of FdU−BdM twin drug nanoparticles was studied with the help of flow cytometry and fluorescence microscopy in HeLa cells. For flow cytometry, 6-well plates were adopted for the seeding of HeLa cells, reaching a cell density of 5.0 × 105 for every well. After that, the cells were cultured with 1.5 mL of complete DMEM and allowed to adhere for 1 day. Diluted solutions of FdU−BdM twin drug nanoparticles and FdU/BdM mixture at a concentration of 40 μM with DMEM culture medium were prepared. After removal of the culture medium, 1.5 mL diluted solutions were put into every well. Then, the cells were incubated for predetermined times (10, 30, 60, and 120 min) at 37 °C. Subsequently, cell growth medium was removed and cells were rinsed with cold PBS three times and treated with trypsin to prepare samples for flow cytometry analysis. The measurement of a BD LSRFortessa flow cytometer was performed to record the data for 1.0 × 104 gated events to analyze cell internalization efficiency. For the fluorescence microscope studies, 6-well plates were adopted for the seeding of HeLa cells, reaching 2.0 × 105 cells for every well in 1.5 mL of complete DMEM. After that, the cells were cultured for 1 day. The culture medium was removed, and the diluted FdU− BdM twin drug nanoparticle solutions with DMEM were added to a concentration of 40 μM. At 37 °C, the cells were continuously cultured for 120 min. After the removal of culture medium, the cold PBS was used for the washing of cells three times. At room temperature, 4% formaldehyde was used to fix the cells for 30 min, and the PBS was used to wash cells three times. Subsequently, 0.1% Triton X-100 solution was used to treat the cells at 0 °C for 15 min, and the cells were washed with PBS three times. Then, the cells were treated with RNase at 37 °C for 20 min and washed with PBS three times. After that, the cells were treated with 1 mL of 2 μg/mL propidium iodide (PI) at room temperature for 15 min. Then, cold PBS was used to wash slides three times, and the slides were dried at room temperature to obtain the resulting slides. Fluorescence microscopy (Olympus Bx60) was performed to mount and observe the resulting slides. Cytotoxicity Measurements of FdU−BdM Twin Drug Nanoparticles. The anticancer activities of FdU−BdM twin drug nanoparticles against HeLa cells, MCF-7 cells, and MCF7/ADR cells were evaluated by MTT viability assay. The free drug FdU, BdM, and the FdU/BdM mixture were used as controls. In the MTT assay, 96-well plates were adopted for the seeding of cells, reaching a cell density of 8 × 103 for every well in 200 μL of culture medium. The cells were incubated for 1 day. After the removal of cell growth medium, 200 μL of a medium containing serial dilutions (FdU−BdM twin drug nanoparticles, free FdU and BdM, or the FdU/BdM mixture from 0.1 to 50 μM for HeLa and MCF-7 cells and from 0.5 to 150 μM for MCF-7/ADR cells) was added. The cells were grown for another 72 h. Then, 20 μL of 5 mg/mL MTT assay stock solution in PBS was added into each well. After

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RESULTS AND DISCUSSION Synthesis and Characterization of FdU−BdM Twin Drug. The FdU−BdM twin drug was prepared by esterification with a molar feed ratio of FdU/BdM at 3:1. The synthetic route is shown in Figure 1. The DCC/DMAP catalytic method was used to make BdM react with FdU to produce the amphiphilic FdU−BdM twin drug. The chemical structure identification data of FdU−BdM twin drug was provided by 1H NMR and 13 C NMR spectroscopy as shown in Figure 2. For the 1H NMR, the peak at 5.13 ppm (1) attributed to hydroxyl proton disappears completely compared with free drug FdU, and the peak at 3.56 ppm (2) related to −CH2− of FdU shifts to 3.58 ppm (2′). Compared with free drug BdM, the 2.38 ppm (3) signal ascribed to methylene (−CH2COOH) of BdM shifts to C

DOI: 10.1021/acs.molpharmaceut.5b00005 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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twin drug (m/z, M + H+) is 586.1649, which is consistent with the calculated value (m/z, M + H+, 586.1637). These experimental results confirm that the FdU−BdM twin drug has been synthesized successfully. The FdU−BdM twin drug was also characterized by FTIR spectroscopy, UV−vis spectrophotometry, and fluorescence spectroscopy. FTIR and UV−vis spectra are shown in Figure S1 in the Supporting Information. In the FdU−BdM FTIR spectrum, the absorption bands at 2363 and 2338 cm−1 disappear completely compared with the FTIR spectrum of BdM. A strong CO stretching absorption band at 1717 cm−1 can be ascribed to the carboxyl stretching vibration. After esterification with FdU, this absorption band moves to 1705 cm−1 due to the formation of the ester linkage. Compared to the FTIR spectrum of FdU, the absorption band at 3312 cm−1 disappears due to the formation of the ester bond. The absorption band at 1267 cm−1 of the FdU−BdM twin drug can be ascribed to the stretching absorption of the bond of C−O−C. In the UV−vis spectrum, the FdU−BdM twin drug possesses both UV−vis absorptions of BdM and FdU. We observe a 5 nm red shift in the absorption of the FdU−BdM twin drug at 270 nm compared to the UV−vis absorption of free FdU at 265 nm. We also observe a slight red shift in the absorption of the FdU−BdM twin drug at 327 nm compared to the UV−vis absorption of free BdM at 324 nm. The fluorescence data are shown in Figure S2 in the Supporting Information. Due to the fluorescence of free BdM, the FdU−BdM twin drug exhibits a fluorescence emission at 420 nm. These experimental results further confirm that the FdU−BdM twin drug has been synthesized successfully. Fabrication of Self-Assembled FdU−BdM Twin Drug Nanoparticles. FdU is a water-soluble anticancer drug, and BdM is a water-insoluble anticancer drug. Therefore, the FdU− BdM twin drug is amphiphilic. The FdU−BdM twin drugs could assemble themselves to form nanoparticles in water benefiting from this amphiphilic nature. The self-assembled FdU−BdM twin drug nanoparticles were prepared by the dialysis method. After adding deionized water into the DMF solution of FdU−BdM twin drug, the DMF was removed by dialysis and a stable nanoparticle solution with a concentration of 0.5 mg/mL was obtained. To confirm whether the nanoparticles formed in water or not, the CAC was estimated by using DPH as a UV probe. DPH shows a higher absorption coefficient in a hydrophobic environment. With increasing concentration of FdU−BdM twin drug nanoparticle solution, the UV absorbance at 313 nm increases. At low FdU−BdM twin drug concentration, the value of absorbance remains nearly unchanged. When the concentration is close to 10 μg/ mL, the absorbance shows a sharp increase. Thus, the UV absorbance curve shows an inflection point. The CAC value of the FdU−BdM twin drug is about 11 μg/mL as given in Figure S3 in the Supporting Information. The CAC value of the FdU− BdM twin drug is close to the CAC value of the nanoparticles formed by the polymers, which demonstrates that the FdU− BdM twin drug nanoparticles possess the advantage of high stability similar to block copolymers. To further confirm that the FdU−BdM twin drug can form nanoparticles, both DLS and TEM measurements were used to study nanoparticles’ size and morphology. The DLS results in Figure 4a show that the FdU−BdM twin drug aqueous solution forms aggregates and the mean hydrodynamic diameter of FdU−BdM twin drug aggregates is about 130.1 nm with a unimodal size distribution. Besides, the DLS measurements were performed at different time intervals. The results demonstrate that the FdU−BdM

Figure 1. Schematic route of FdU−BdM twin drug and construction of self-assembled nanoparticles for cancer therapy.

Figure 2. (a) 1H NMR and (b) 13C NMR spectra of BdM, FdU, and FdU−BdM twin drug in DMSO.

2.39 ppm (3′). In Figure 2b, compared with the 13C NMR spectrum of BdM, the 174.64 ppm signal from −COOH (1) shifts to 172.92 ppm (1′), and the 32.96 ppm signal from the methylene (−CH2COOH) (2) moves to 33.07 ppm (2′). Furthermore, the 70.80 ppm (3) and 88.15 ppm (4) signals from the methylene (−CH2−CH−) of FdU move to 70.45 ppm (3′) and 85.02 ppm (4′). To verify the molecular weight and purity of FdU−BdM twin drug, the LC−MS technique was performed, and the results are shown in Figure 3. LC profile gives one retention time of the FdU−BdM twin drug at 3.15 min, indicating the high purity of the FdU−BdM twin drug. The MS data show that the molecular weight of the FdU−BdM

Figure 3. Mass spectrum of FdU−BdM twin drug. Inset: The LC profile of FdU−BdM twin drug. D

DOI: 10.1021/acs.molpharmaceut.5b00005 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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concentration difference between the solutions inside and outside the dialysis bag at the beginning of the release. With the increase of time, the concentration difference decreases and the concentration inside and outside the dialysis bag reaches equilibrium finally. Therefore, the cumulative release of BdM would reach a plateau. The release of hydrophilic floxuridine was further studied, and the release profiles of hydrophilic floxuridine from the nanoparticles are presented in Figure S5 in the Supporting Information. The profiles demonstrate that the release trend of hydrophilic floxuridine is similar to that of the hydrophobic bendamustine, but the release of floxuridine is faster than that of bendamustine because the bendamustine molecules are encapsulated within the micelles after the cleavage of ester bond considering the hydrophobic interactions. To further confirm whether the FdU−BdM twin drug was converted into free FdU and BdM in cells or not, we evaluated the intracellular degradation of FdU−BdM twin drug. After the HeLa cancer cells were incubated with FdU−BdM twin drug nanoparticles for 6 h, we used the LC−MS technique to measure the cellular extracts. The LC−MS data in Figure S7 in the Supporting Information show that the peaks of free FdU and BdM and FdU−BdM twin drug were displayed in the cellular extracts after incubation for 6 h with FdU−BdM twin drug nanoparticles. The results demonstrate that the FdU− BdM twin drug nanoparticles could enter into cancer cells and the ester bond between FdU and BdM is readily cleaved by hydrolysis to release two active principles within cancer cells. Cell Internalization. To determine whether the FdU− BdM twin drug nanoparticles could effectively deliver drugs into cells or not, the cellular uptake of FdU−BdM twin drug nanoparticles by HeLa cells was evaluated by fluorescence microscopy and flow cytometry. The fluorescence spectra show that BdM can emit blue fluorescence, and the self-assembled FdU−BdM twin drug nanoparticles also exihibit strong blue fluorescence in water. The fluorescence spectra of BdM, FdU− BdM twin drug, and FdU−BdM twin drug nanoparticles are given in Figures S2a and S2b in the Supporting Information. Due to the inherent fluorescence of BdM, the FdU−BdM twin drug itself could be used as a probe for the cell internalization analysis. For the flow cytometric analysis, the HeLa cells were cultured at 37 °C for 10 min, 30 min, 60 min, and 120 min after the FdU−BdM twin drug nanoparticles and FdU/BdM mixture with a concentration of 40 μM were added to culture medium. The relationship of the fluorescence intensity of cells and incubation time is shown in Figure 6. With the increase of incubation time, the fluorescence intensity gradually increases, which indicated that more and more FdU−BdM twin drug nanoparticles enter into HeLa cells and the FdU−BdM twin drug nanoparticles exhibit effective cellular uptake. To further study the cell internalization efficiency between FdU−BdM twin drug and FdU/BdM mixture, the cell internalization of FdU/BdM mixture has been investigated. Compared to the uptake curve of FdU−BdM twin drug nanoparticles as shown in Figure S8 in the Supporting Information, the cell internalization efficiency of FdU/BdM mixture is more effective in 2 h. The reason is that these two types of drug formulas enter into cells in two different ways. For FdU/BdM mixture, drug molecules enter into cells by diffusion, which will be faster than the endocytosis way for nanoparticles in a short time. The cellular uptake of FdU−BdM twin drug nanoparticles was further studied by fluorescence microscopy. HeLa cells were treated with FdU−BdM twin drug nanoparticles and

Figure 4. (a) DLS plot of FdU−BdM twin drug nanoparticles, which shows the average size (Dh = 130.1 nm) and the polydispersity index (PDI = 0.15). Inset: a digital photograph of transparent bluish FdU− BdM twin drug nanoparticle solution. (b) TEM image of FdU−BdM twin drug nanoparticles. (Count: n = 60.)

twin drug nanoparticles are stable enough for at least one-week storage (Figure S4 in the Supporting Information). TEM technique was used to further measure and visualize the size and morphology of the aggregates. The TEM image in Figure 4b shows that the FdU−BdM twin drug aggregates into approximate spherical micelles in aqueous solution, and the size determined by TEM is about 118.9 ± 8.1 nm (n = 60), which is close to that measured by DLS. These results demonstrated that FdU−BdM twin drug can self-assemble into stable and well-defined nanoparticles. In Vitro FdU and BdM Release. The in vitro release behavior of FdU−BdM twin drug nanoparticles was investigated under a simulated physiological condition (PBS, pH = 7.4) and in an acidic environment (acetate buffer, pH = 5.0) containing (or not) esterase (30 U/mL) at 37 °C. Figure 5

Figure 5. In vitro BdM release kinetics from FdU−BdM twin drug nanoparticles at different pH values (5.0 and 7.4) containing esterase (or not) at 37 °C.

presents the release curves of BdM from the nanoparticles at different conditions. The FdU−BdM twin drug nanoparticles release less than 20% of BdM in pH = 7.4 PBS solution over a period of 48 h, which demonstrates that the FdU−BdM twin drug nanoparticles exhibit high stability in the physiological condition. When pH = 5.0, more free BdM drugs are released. It is worth noting that the existence of esterase promotes the release of BdM, and the release rate is faster than that under the physiological and acidic conditions. Especially, the FdU−BdM twin drug nanoparticles release 50% of BdM in an acidic environment containing esterase, because the esterase and acidic condition cause the quick degradation of the ester bond. In Figure 5, the release of BdM is rapid at the early stage and then reaches a plateau after 15 h, because there is a large E

DOI: 10.1021/acs.molpharmaceut.5b00005 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 8. Cell viability of (a) HeLa cells and (b) MCF-7 cells incubated with FdU, BdM, FdU/BdM mixture, and FdU−BdM twin drug nanoparticles after 72 h at various concentrations determined by MTT assay. Values are presented as average ± standard error (n = 3).

Figure 6. Cellular uptake of FdU−BdM twin drug nanoparticles by HeLa cells. (a) The relationship of fluorescence intensity and time of FdU−BdM twin drug nanoparticle in the HeLa cells by flow cytometry analysis. (b) Representative flow cytometry histogram profiles of HeLa cells without any treatment. (c) Representative flow cytometry histogram profiles of HeLa cells cultured with FdU−BdM twin drug nanoparticles for 2 h.

cells is higher than that of free FdU and FdU/BdM mixture at higher concentration range, because when the concentration is above the CAC value, the amphiphilic twin drug self-assembles into nanoparticles. The FdU−BdM twin drug nanoparticles would accumulate in the tumor cells because of cellular uptake, and a synergistic action might be displayed by the two released free drugs. It is found that a similar phenomenon also appeared in the MCF-7 cells, while the cytotoxicity to MCF-7 cancer cells is higher than that to HeLa cells. The reason is that both FdU and BdM are used for the treatment of breast cancer. Multidrug Resistance. The development of multidrug resistance (MDR) is one of the major obstacles to effective cancer chemotherapy, whereby cancer cells become resistant to the cytotoxic effects of various chemotherapeutic agents. The current strategies to overcome the tumor MDR include nanotechnologies, multidrug combined chemosensitization, and the reconstruction of primary drugs.33 Nanotechnology is a valuable candidate approach to overcome MDR due to the advantages including enhancing the accumulation and internalization of drugs within tumors, stimulus-sensitive intracellular release, simultaneous targeting delivery of different agents, and the findings of “pharmacologically active” nanocarrier materials.34 The synthesized FdU−BdM twin drug nanoparticles have two different chemotherapeutic agents. Hence, it is expected that FdU−BdM twin drug nanoparticles would be potential candidates to reverse the MDR of cancer cells. To confirm whether the FdU−BdM twin drug nanoparticles would be exhibit higher anticancer efficiency on MDR tumor cells than free drugs and the mixture of free drugs, we investigated the cytotoxicity of BdM, FdU, FdU/BdM mixture, and FdU−BdM twin drug nanoparticles by MTT assay (Figure 9). As shown in Figure 9, the IC50 values of free FdU and FdU/BdM mixture are 100 and 70 μM in the MCF-7/ADR cells, individually, which is approximately 25-fold of the IC50 value in the MCF-7

cultured for 2 h before observation, and PI was used to stain the nuclei for 15 min. As shown in Figure 7, both cytoplasm and

Figure 7. Cellular uptake of FdU−BdM twin drug nanoparticles by HeLa cells using fluorescence microscopy to observe. (a) Fluorescence microscopy photos of HeLa cells incubated with FdU−BdM twin drug nanoparticles for 2 h. (b) PI stained the cell nuclei. (c) The blue fluorescence of FdU−BdM twin drug nanoparticles in cells. (d) The merged image demonstrates that the FdU−BdM twin drug nanoparticles are in both cytoplasm and nuclei.

nuclei of the cells treated with FdU−BdM twin drug nanoparticles exhibit blue fluorescence according to the merged image, which demonstrates that FdU−BdM twin drug nanoparticles could enter into cancer cells in 2 h. It is worth noting that the FdU−BdM twin drug can arrive at the nuclei in a shorter time than the free drugs encapsulated in the polymer nanoparticles.30−32 In Vitro Cytotoxicity of FdU−BdM Twin Drug Nanoparticles. The proliferation inhibition of FdU−BdM twin drug nanoparticles compared with free FdU and BdM and FdU/ BdM mixture was evaluated against HeLa and MCF-7 cancer cells under identical conditions by MTT assay. The cells were treated with the nanoparticles and free drugs at different concentration from 0.125 to 50 μM. The cells without any treatment were used as the control. The cell proliferation results are shown in Figure 8. After incubation for 72 h, the cytotoxicity of FdU−BdM twin drug nanoparticles to cancer

Figure 9. Cell viability of MCF-7/ADR cells cultured with FdU, BdM, FdU/BdM mixture, and FdU−BdM twin drug nanoparticles determined by MTT assay after 72 h. F

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Figure 10. Cell apoptosis data of HeLa cells treated with FdU, BdM, FdU/BdM mixture, and FdU−BdM twin drug nanoparticles for 24 h by flow cytometry analysis. Inserted numbers in the profiles present the percentage of the cells in this area. Lower left: living cells. Upper left: necrotic cells. Lower right: early apoptotic cells. Upper right: late apoptotic cells.

cells (IC50: 4 μM for FdU, 3 μM for FdU/BdM mixture; Table S1 in the Supporting Information). However, the IC50 (15 μM) of FdU−BdM twin drug nanoparticles in MCF-7/ADR cells is just 5-fold of that (3 μM) in MCF-7 cells. According to the IC50 values, the resistance index (RI) can be calculated. The RI of FdU and FdU/BdM mixture is 25 and 23, respectively. But for the FdU−BdM twin drug nanoparticles, the RI is just 5. The data demonstrate that the FdU−BdM twin drug nanoparticles can be applied to overcome the MDR. Furthermore, the amphiphilic twin drug strategy provides a new platform for overcoming the MDR and, thus, can lead to an excellent anticancer activity. Apoptosis of Cancer Cells Induced by FdU−BdM Twin Drug Nanoparticles. Apoptosis is the principal mechanism of tumor cell death by anticancer drugs.35 Here, to confirm whether the cancer cell death caused by FdU−BdM twin drug was associated with apoptosis or not, the FITC−annexin V/ propidium iodide (PI) method was used to measure the ratio of apoptosis cells. HeLa cells were incubated with FdU, BdM, FdU/BdM mixture, and FdU−BdM twin drug nanoparticles, and the concentration is 20 μM, respectively. After 24 h incubation, FITC−annexin V/PI was used to stain the cells. The cells without any treatment were used as control. Flow cytometry data are shown in Figure 10. The ratio of apoptosis cells is 15.7%, 61.1%, 65.3% ,and 71.6% induced by FdU, BdM, FdU/BdM mixture, and FdU−BdM twin drug nanoparticles. The data demonstrate that that FdU−BdM twin drug could induce the cell apoptosis and the FdU−BdM twin drug nanoparticles promote higher apoptotic rate of HeLa cells in comparison with other formulations with the same concentration. Caspase-3 Protein Activity Assay. It is known that cell apoptosis requires caspases activated by various stimuli. Among them, caspase-3 protease is considered to be very important in signal transforming of cell apoptosis, and it was reported that caspase-3 could be induced by cytotoxic drugs.36 To determine whether the caspase-3 was activated by FdU−BdM twin drug nanoparticles or not, the expression of caspase-3 protein was evaluated using the substrate of Ac-DEVD-pNA. The HeLa cells were treated with FdU, BdM, FdU/BdM mixture, and FdU−BdM twin drug nanoparticles for 24 h at a concentration of 20 μM; and for MCF-7/ADR cells, the concentration is 40 μM. The untreated cells were used as control. Figure 11 demonstrates that, when HeLa and MCF-7 cells are treated with BdM, FdU, and FdU/BdM mixture, the caspase-3 protein expression increases slightly in comparison with the control.

Figure 11. (a) Caspase-3 protein activity in HeLa cells (a), MCF-7 cells (b), and MCF-7/ADR cells (c) activated by FdU, BdM, FdU/ BdM, and FdU−BdM twin drug nanoparticles at a concentration of 20 μM for HeLa cells and MCF-7 cells and 40 μM for MCF-7/ADR cells.

However, when the HeLa and MCF-7 cells are treated with FdU−BdM twin drug nanoparticles, the expression of caspase-3 protein is upregulated markedly. Although caspase-3 can be activated by BdM, FdU, and FdU/BdM mixture, the results clearly indicate that the FdU−BdM twin drug nanoparticle is the most effective activator to increase the caspase-3 activity. To further confirm that the caspase-3 was activated by FdU− BdM twin drug nanoparticles in MCF-7/ADR cells, we studied the caspase-3 protein activity in MCF-7/ADR cells. The cells were treated with FdU−BdM twin drug nanoparticles and free drugs at the same concentration (40 μM) for 24 h, and cells without any treatment were used as control. It was found that caspase-3 protein activity in MCF-7/ADR cells treated with FdU−BdM twin drug nanoparticles increased more than that treated with free drugs. These results demonstrate that the selfassembled FdU−BdM twin drug is an effective strategy to activate the expression of caspase-3 in MCF-7/ADR cells, which also indicates that the self-assembly of amphiphilic FdU−BdM twin drug is a valuable candidate approach to overcome MDR. G

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CONCLUSION To summarize, we put forward a strategy to fabricate twin drug nanostructures with high and fixed drug loading of anticancer drugs for cancer therapy. In this study, the FdU−BdM twin drug can self-assemble into stable and well-defined nanoparticles in the aqueous solution. Benefiting from their nanoscale characteristics, FdU−BdM twin drug nanoparticles can overcome MDR of the tumor cells. After hydrolysis of the FdU−BdM twin drug nanoparticles in the cells, both FdU and BdM can be released as active molecules to kill cancer cells, resulting in a better anticancer efficacy, because of higher expression of caspase-3 and apoptotic rate than the free drugs. We believe that this amphiphilic twin drug strategy can be extended to construct nanostructures of other hydrophilic and hydrophobic anticancer drugs, and it should be possible to form nanoparticles under proper self-assembly conditions. It is expected that this strategy will open a new way for the development of a drug self-delivery system due to the small molecular anticancer drugs being used as active building units in the nanoparticles, not just passive cargoes to be delivered.

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ASSOCIATED CONTENT

S Supporting Information *

The synthesis details and characterizations of FdU−BdM twin drug and FTIR, UV−vis, and fluorescence characterization data for the FdU−BdM twin drug; preparation of FdU−BdM twin drug nanoparticles; in vitro cell experiments. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00005.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +86-21-54741297. *E-mail: [email protected]. Fax: +86-21-54741297. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Basic Research Program (2015CB931801, 2013CB834506, 2012CB821500) and National Natural Science Foundation of China (51473093). We thank Lei Feng (Instrumental Analysis Center of Shanghai Jiao Tong University) for LC−MS assay.

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REFERENCES

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