Enhanced effect of pH-sensitive mixed copolymer micelles for overcoming multidrug resistance of doxorubicin

Biomaterials xxx (2014) 1e11

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Enhanced effect of pH-sensitive mixed copolymer micelles for overcoming multidrug resistance of doxorubicin Lipeng Qiu a, b, 1, Mingxi Qiao b, 1, Qing Chen b, Chenmin Tian c, Miaomiao Long b, Mingyue Wang b, Zhen Li b, Wen Hu c, Gang Li b, Liang Cheng c, Lifang Cheng c, Haiyang Hu b, Xiuli Zhao b, Dawei Chen b, c, * a b c

Department of Pharmaceutics, School of Pharmaceutical Sciences, Jiangnan University, No. 1800, Lihu Road, Wuxi 214122, PR China Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, PR China Department of Pharmaceutics, College of Pharmaceutical Science, Soochow University, No. 199, Renai Road, Suzhou 215123, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 July 2014 Accepted 1 August 2014 Available online xxx

P-glycoprotein (P-gp) mediated drug efflux has been recognized as a key factor contributing to the multidrug resistance (MDR) in tumor cells. To address this issue, a new pH-sensitive mixed copolymer micelles system composed of hyaluronic acid-g-poly(L-histidine) (HA-PHis) and D-a-tocopheryl polyethylene glycol 2000 (TPGS2k) copolymers was developed to co-deliver doxorubicin (DOX) and TPGS2k into drug-resistant breast cancer MCF-7 cells (MCF-7/ADR). The DOX-loaded HA-PHis/TPGS2k mixed micelles (HPHM/TPGS2k) were characterized to have a unimodal size distribution, high DOX loading content and a pH dependent drug release profile due to the protonation of poly(L-histidine). As compared to HA-PHis micelles (HPHM), the HPHM/TPGS2k showed higher and comparable cytotoxicity against MCF-7/ADR cells and MCF-7 cells, respectively. The enhanced MDR reversal effect was attributed to the higher amount of cellular uptake of HPHM/TPGS2k in MCF-7/ADR cells than HPHM, arising from the inhibition of P-gp mediated drug efflux by TPGS2k. The measurements of P-gp expression level and mitochondrial membrane potential indicate that the blank HPHM/TPGS2k inhibited P-gp activity by reducing mitochondrial membrane potential and depletion of ATP but without inhibition of P-gp expression. In vivo study of micelles in tumor-bearing mice indicate that HPHM/TPGS2k could reach the tumor site more effectively than HPHM. The pH-sensitive mixed micelles system has been demonstrated to be a promising approach for overcoming the MDR. © 2014 Published by Elsevier Ltd.

Keywords: pH-sensitive Copolymer micelles Multidrug resistance Hyaluronic acid D-a-tocopheryl polyethylene glycol succinate P-glycoprotein

1. Introduction Doxorubicin (DOX), an antibiotic belonging to the class of anthracyclines, is one of the most important cytotoxic drug used in treating breast cancer [1]. However, two factors severely compromised the therapeutic efficacy of DOX. One is the cardio-toxicity arising from the side effect of DOX due to its distribution in heart [2]. Another is the development of multidrug resistance (MDR) during the chemotherapy [3]. The cardio-toxicity of DOX can be circumvented by developing a nanoparticle based targeting delivery system so as to reduce the distribution in heart. However,

* Corresponding author. Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, PR China. Tel./fax: þ86 512 65884729. E-mail address: [email protected] (D. Chen). 1 Both authors contributed equally to this work.

lack of effective approach to overcome MDR still remains a primary challenge for DOX and other anticancer drugs despite much effort has been made to tackle it in the last decade [4,5]. Although there are several different mechanisms associated with the development of MDR, the primary cause has been recognized to be the overexpression of ATP binding cassette (ABC) transporters, particularly P-glycoprotein (P-gp), leading to the efflux of anticancer drugs [6]. Many attempts have been made to deliver anticancer drugs to MDR cells using various nanoparticles due to the characteristics of evading P-gp efflux by receptormediated endocytosis [7,8]. For example, Kim et al. developed a targeted polymeric micelles in an attempt to overcome tumor MDR [9]. The micelles showed enhanced cytotoxicity against MDR cells due to the folate receptor-mediated endocytosis that evaded the Pgp mediated efflux. However, the approach to bypass the P-gp mediated efflux showed limited MDR reversal due to no inhibition effect on P-gp transporter. Despite the nanoparticles were capable

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of delivery the incorporated drugs into cytoplasm of cancer cells by evading P-gp mediated efflux, P-gp transporters still removes the drugs from cells before they access to the site of drug action. Therefore, much effort has been devoted to develop nanoparticles based drug delivery systems that suppressed the function of P-gp to reverse MDR [10]. In order to inhibit P-gp function, a broad range of compounds that interact with P-gp and block drug efflux have been used to reverse the MDR phenotype. However, their applications were hindered by the inherent toxicity, low efficacy or the altered pharmacokinetics and toxicity of cytotoxic drugs [11,12]. Recently, various amphiphilic copolymers, such as D-a-tocopheryl polyethylene glycol 1000 succinate (TPGS) and pluronics have been identified to be the most promising P-gp inhibitors due to less concern on safety issues than their small molecular inhibitors [13]. TPGS has been demonstrated to inhibit the efflux pumps, leading to the restored sensitivity to anticancer drugs [14]. By investigating TPGS and its analogs that varied by their PEG chain length, or possessed a modified hydrophobic core, some studies have proved that P-gp ATPase inhibition in the inhibitory mechanism is an essential factor on cellular efflux pump [15e17]. Moreover, TPGS is widely used for synthesis of biodegradable copolymers, resulting in high drug encapsulation efficiency, high cellular uptake and therapeutic effects in vitro and in vivo [14,18,19]. Therefore, a number of TPGS-based copolymer micelles have been developed in an attempt to overcome tumor MDR [20e22]. For example, TPGS/PLGA nanoparticles were developed to reverse tumor MDR. The cytotoxicity of TPGS/PLGA nanoparticles against A549/DDP MDR cells was found to increase 3.56 times as compared to that of free drug [23]. The mixed micelles composed of TPGS and poly(D,L-lactide-co-glycolide)-poly(ethylene glycol)-folate (PLGA-PEG-FOL) showed higher cellular uptake by DOX-resistant KB cells and subsequent higher cytotoxicity than PLGA-PEG-FOL micelles [24]. However, the relatively high critical micelle concentration (0.2 mg/ml) of TPGS may compromise the stability of the mixed micelles in physiological environment, resulting in the leak of TPGS from the micelles before reaching the target. Furthermore, the chain length of PEG1000 in TPGS is not long enough to ensure the extended blood circulation of the micelles in blood circulation [25]. Moreover, one major concern with the previously developed micelles is whether the micelles are capable of effectively delivering TPGS into the cytosol after being taken up and encapsulated in the endosomes/lysosomes (endo-lysosomes) of the MDR cells. To address the above mentioned issues, a TPGS analog consisting of PEG2000 (TPGS2k) and hyaluronic acid-g-poly(L-histidine) (HA-PHis) were used to construct a new mixed micellar system with endo-lysosomal escape property for intracellular co-delivery of the drugs and TPGS2k to MDR cells. Hyaluronic acid (HA) is a biodegradable polyanionic polysaccharide with low toxicity, and more importantly a ligand for CD44 receptors overexpressed in a variety of cancer cells [26,27]. Poly(L-histidine) (PHis) is a pH sensitive polymer with endo-lysosomal escape property [28]. The HAPHis micelles are expected to integrate the CD44 receptor mediated active targeting, pH triggered payload release and endo-lysosomal escape property. The micelles will provide an effective approach for bypassing P-gp efflux by increasing cellular uptake and rapid delivery of the cargoes into the cytosol. The TPGS2k was introduced into mixed micelles to ensure long blood circulation due to the relatively long PEG block and to inhibit the P-gp efflux pump for overcoming the tumor MDR. The mixed micelles are expected to passively accumulate in the tumor tissue by EPR effect, and to be internalized into the tumor cells via CD44 receptor-mediated endocytosis. After endocytosis, the mixed micelles disassemble in the acidic end-lysosomes due to protonation of PHis block, resulting in the burst release of DOX and TPGS2k. The PHis facilitate the

endo-lysosomal escape and translocation of DOX and TPGS2k to the cytosol, achieving the MDR reversal effect (Fig. 1). In this study, the HA-PHis and TPGS2k polymers were synthesized respectively and used to prepare the mixed micelles. The characteristics of the mixed micelles such as drug loading, particle size and in vitro drug release were tested. The cellular uptake and reversal of MDR by DOX-loaded mixed micelles were investigated with MCF-7/ADR cells. Moreover, the effect of the mixed micelles on the P-gp expression and the P-gp activity related influencing factors, such as intracellular ATP level and mitochondrial membrane potential were also investigated. 2. Experimental section 2.1. Materials Sodium hyaluronate (MW 11000) was purchased from Shandong Freda Biopharm (Shandong, China). Na-CBZ-Nim-DNP-L-histidine was purchased from GL Biochem (Shanghai, China). D-a-tocopheryl acid succinate and methoxyl PEG amine, MW 2000 (mPEG2k-NH2) were bought from Jiangsu Xixin International Co., Ltd. (Suqian, China) and Yare Biotech (Shanghai, China) respectively. Nhydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Doxorubicin Hydrochloride was purchased from Huafeng United Technology (Beijing, China). Pyrene and rhodamine 123 (Rh123) were supplied by SigmaeAldrich (MO, USA). Rabbit anti-P-gp was purchased from biosynthesis biotechnology (Beijing, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Hoechst 33342, JC-1, ATP assay kit, BCA protein assay kit and FITC-labeled goat anti-rabbit IgG were all bought from Beyotime (Shanghai, China). Dulbecco's modified Eagle medium (DMEM), RPMI 1640 medium, fetal bovine serum (FBS) and penicillinestreptomycin solution were purchased from Gibco BRL (Maryland, USA). All the other chemicals and buffer solution components were analytical grade.

2.2. Cell cultures Human breast carcinoma Michigan Cancer Foundation-7 cells (MCF-7) and DOXresistant MCF-7 cells (MCF-7/ADR) were provided by Chinese Academy of Sciences (Shanghai, China) and KeyGen Biotech Co. Ltd (Nanjing, China) respectively. MCF7 cells were cultured in DMEM supplemented with 10% of FBS and 1% penicillinestreptomycin solution. MCF-7/ADR cells were maintained in RPMI 1640 medium containing 10% FBS, 1% penicillinestreptomycin solution and 1000 ng/mL DOX. All cells were cultured at 37  C in a humidified atmosphere with 5% CO2. 2.3. Synthesis and characterization of HA-PHis and TPGS2k copolymers 2.3.1. Synthesis of HA-PHis HA-PHis was synthesized as described in our previous publication [29]. Briefly, HA (0.26 mmoL) was dissolved in 10 mL of anhydrous formylamine at 40  C. After the solution was cooled to room temperature, EDC (0.52 mmoL) and NHS (0.52 mmoL) were added and stirred for 2 h in ice bath. Then PHis-DNP (0.52 mmoL) was dissolved in anhydrous N,N-dimethylformamide and added slowly to the HA solution. The reaction mixture was stirred at 50  C for 6 h and further for additional 40 h at room temperature under nitrogen atmosphere. The resulting solution was dialyzed with distilled water/ethanol (1/3 e 1/1, v/v) for 1 day and distilled water for 2 days, respectively [30]. The solution was filtered to remove the precipitates and freeze-dried to obtain HA-PHis-DNP powder. The HA-PHis-DNP powder was dissolved in 5 mL of formylamine followed by adding b-mercaptoethanol. The mixture was stirred for 24 h at 30  C under nitrogen atmosphere to cutoff the DNP group. After the reaction, the solution was dialyzed against distilled water for 3 days followed by freeze drying to obtain HA-PHis powder. 2.3.2. Synthesis of TPGS2k The synthesis of TPGS2k was carried out as described previously with brief modification [14]. Briefly, D-a-tocopheryl succinate (VES) and mPEG2k-NH2 were dissolved in dichloromethane followed by adding EDC and NHS with stoichiometric ratio of 1:1:2:2. The mixture was stirred at 30 C for 48 h under nitrogen atmosphere in dark. After reaction, the solution was filtered and then precipitated in cold diethyl ether. The obtained precipitate was dissolved in water and dialyzed against distilled water for 3 days. The solution was filtered to remove impurities and freeze-dried to obtain TPGS2k powder. 2.3.3. Characterization of HA-PHis and TPGS2k The chemical structure of HA-PHis and TPGS2k was confirmed by 1H NMR spectrum recorded on Varian Mercury Plus-400 NMR spectrometer (Varian, USA) operating at 400 MHz respectively.

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Fig. 1. Schematic illustration of the approach to overcome MDR by DOX-loaded HA-PHis/TPGS2k mixed copolymer micelles (HPHM/TPGS2k). HPHM/TPGS2k is selectively uptaken by DOX-resistant MCF-7 cells via CD44 receptor-mediated endocytosis to evade P-gp efflux, and pH-triggered released of DOX and TPGS2k into the cytoplasm to suppress P-gp efflux pump and improve the intracellular drug accumulation.

2.3.4. Measurement of CMC values of HA-PHis and TPGS2k The critical micelle concentration (CMC) was determined using a fluorescence spectrophotometer with pyrene as a probe [31]. A stock solution of pyrene (6.0  105 M) was prepared in acetone and dropped into the brown volumetric flask. The acetone was completely evaporated under a gentle nitrogen gas stream for 1 h at 60  C. HA-PHis solution (concentration ranging from 5.0  104 to 2.0 mg/mL) and TPGS2k (concentration ranging from 1  103 to 10 mg/mL) was added to each volumetric flask to achieve a final pyrene concentration of 6.0  107 M, respectively. The fluorescence intensity was measured using a fluorescence spectrophotometer (LS55, PerkinElmer, USA) and an emission spectrum (350e450 nm) was obtained at a fixed excitation wavelength of 336 nm. The intensity ratio of the first peak (I1, 373 nm) to the third peak (I3, 384 nm) in the emission spectrum was analyzed for calculation of the CMC. 2.3.5. Determination of hemolytic activity of HA-PHis and TPGS2k The hemolytic activity of HA-PHis and TPGS2k polymers was investigated using rabbit red blood cells (RBC) with Tween 80 used as a control [32]. Briefly, RBC suspension (0.5 mL) was added to 0.5 mL of samples to make the concentrations of HA-PHis, TPGS2k, and Tween 80 ranging from 10 to 500 mg/mL. The samples were incubated at 37  C for 2 h and then centrifuged at 5000 rpm for 5 min to remove intact RBC. The supernatant was analyzed for released hemoglobin with a UV-2600 spectrophotometer (Shimadzu, Japan) at 570 nm. Normal saline and distilled water were added to obtain 0 and 100% hemolysis, respectively. The degree of hemolysis was calculated by the following equation.
As  A0 Hemolysis % ¼ A100  A0

 100

where, As is the absorbance of a sample, A100, and A0 are the absorbance of the reference solutions at 100% hemolysis and 0% hemolysis respectively. 2.4. Preparation of DOX-loaded copolymer micelles DOX-loaded HA-PHis/TPGS2k (HPHM/TPGS2k) mixed micelles were prepared by probe-type ultrasonication technique [29]. HA-PHis (10 mg) and TPGS2k (2 mg) were dissolved in 10 mL of PBS (0.1 M, pH 8.0). DOX (2 mg) was dissolved in 4 mL tetrahydrofuran/acetone (1:1, v/v). The DOX solution was slowly injected into copolymer PBS solution with stirring at room temperature. The mixture was ultrasonicated for 30 min (active every 2 s for a 3 s duration with an output power of 100 W, JY92-II, Xinzhi Scientific Co., Ltd., Ningbo, China) in ice bath. The mixture was stirred at room temperature for 24 h to remove the organic solvent. The obtained micellar dispersion was filtered through a 0.45 mm membrane to eliminate insoluble drug. The DOX-loaded HA-PHis (HPHM) micelles and the blank HA-PHis-based micelles were prepared as described above without adding TPGS2k and DOX respectively.

2.5. Characterization of DOX-loaded copolymer micelles 2.5.1. Particle size, size distribution and zeta potential The particle size, size distribution and zeta potential of the mixed micelles were assessed using a Zeta Potential/Particle Sizer Nicomp™ 380ZLS (PSS$Nicomp, Santa Barrara, USA). 2.5.2. Transmission electron microscopy (TEM) The morphology of the mixed copolymer micelles was observed using a transmission electron microscopy (TEM) (TecnaiG220, FEI, USA). Before visualization, the sample was placed on copper grids with films, air-dried for 10 min and finally examined through TEM with an accelerated voltage of 120 KV. 2.5.3. The encapsulation efficiency (EE) and drug loading content (DL) The EE and DL of the mixed micelles was measured with UV-spectrophotometer (UV-2600, Shimadzu, Japan) at 479 nm respectively. Briefly, DOX-loaded mixed micelles was diluted 10-fold by formylamine and then ultrasonic for 10 min to collapse nanoassemblies and to dissolve the DOX. The concentration of DOX was determined by a UV/visible spectrophotometer with the wavelength at 479 nm. The DOX content was calculated from the calibration curve over the linear range from 1 to 32 mg/mL. The EE of DOX was calculated as the ratio of the mass of drug encapsulated in micelles to the mass of DOX added. The DL was calculated as the percentage of the mass of DOX encapsulated in micelles to the mass of the DOX-loaded micelles. 2.6. In vitro pH-sensitive release of DOX from micelles The in vitro release of DOX from the mixed micelles was investigated using a dialysis bag (molecular weight cutoff (MWCO) 3500 Da) at 37  C under sink conditions. Briefly, 2.0 mL of DOX-loaded mixed micelles was introduced into a dialysis bag. The dialysis bag was immersed in 20 mL of fresh PBS solution (pH 5.0 and 7.4, 0.1 M) in a vial, which was placed in a shaking incubator at a stirring speed of 100 rpm at 37  C. At predetermined time internals, a 1.0 mL sample was taken from the release medium and the same volume of fresh buffer was added to maintain the volume. The concentration of DOX released into PBS was measured using a UV/ visible spectrophotometer with the wavelength at 479 nm as described above. 2.7. In vitro cytotoxicity assays The cytotoxicity of blank micelles and DOX-loaded micelles against MCF-7 and MCF-7/ADR cells was evaluated by the standard MTT assay [33]. Briefly, the cells were seeded at the density of 7  103 cells/well in 96-well plates and incubated for 24 h to allow cell attachment. The cells were then incubated with free DOX, blank micelles and DOX-loaded micelles at 37  C. After 48 h of incubation, 10 mL of MTT (5 mg/mL) was added to medium and further incubated for 4 h. Afterwards, the

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medium in each well was then removed and 100 mL of DMSO was added to dissolve the internalized purple formazan crystals. The absorbance at 492 nm was recorded using a BioRed microplate reader (MK3, Thermo, USA). The relative cell viability (%) was calculated using the following equation. Cell viability %




Asample  Ablank ¼  100 Acontrol  Ablank

where, Acontrol and Asample are the absorbance in the absence and in the presence of sample treatment respectively, and Ablank is the absorbance of the medium. The IC50 value (the concentration that inhibited cell growth by 50%) was calculated using SPSS 17.0 (Chicago, IL, USA). The resistant index (RI) and reversal factor (RF) were calculated to evaluate the MDR reversal effect of the micelles. RI ¼ IC50

(MCF-7/ADR)/IC50 (MCF-7)

RF ¼ IC50

(DOx)/IC50 (Copolymer micelles)

2.8. Cellular uptake The cellular uptake of micelles was analyzed quantitatively using flow cytometry. MCF-7 and MCF-7/ADR cells were seeded on a 6-well culture plate at the density of 3  104 cells per well for 24 h. After adding free DOX and DOX-loaded micelles with equivalent DOX concentration (5.0 mg/mL), cells were incubated for another 2 h. Then, the cells were washed three times with ice-cold PBS (pH 7.4) and harvested. The cells were resuspended in 0.5 mL of PBS and analyzed using a flow cytometer (FACSCalibur, BD Biosciences, USA). 2.9. Intracellular pH-triggered release of DOX Confocal laser scanning microscopy (CLSM) was used to determine the subcellular localization and intracellular release of DOX. The MCF-7/ADR cells were cultured on microscope slides in a 6-well plate (1  104 cells/well) and incubated for 24 h. The cells were washed and incubated with the micelles for a given time at 37  C. Then cells were treated with LysoTracker green (80 mg/mL) for 60 min and Hoechst 33342 (10 mg/mL) for 20 min to visualize endo-lysosomes and nucleus respectively. The cells were washed three times with ice-cold PBS and mounted on a slide with 5% paraformaldehyde for CLSM (LSM710, ZEISS, Germany) observation. In order to study the effect of the endo-lysosomal pH on the drug release from the micelles, cells were pre-incubated with 8 mg/mL of chloroquine for 1 h before they were exposed to the micelles solution [34]. 2.10. Rhodamine 123 (Rh123) accumulation and efflux The accumulation and efflux of Rh123 were taken as an index of P-gp activity [35,36]. Briefly, MCF-7 and MCF-7/ADR cells were seeded into 6-well plates at a density of 3  104 cells/well. The cells were exposed to Rh123 in blank micelles and free Rh123 solution at same concentration (5.0 mM). After 2 h incubation, the cells were washed and resuspended in 0.5 mL PBS. The intracellular accumulation of Rh123 was detected using a flow cytometer (FACSCalibur, BD Biosciences, USA). For Rh123 efflux assay, cells were pretreated with 5.0 mM Rh123 for 1 h. After the pretreatment, cells were incubated with culture medium and blank micelles for another 1 h. Then the cells were washed and the Rh123 content was analyzed using the above method. 2.11. P-gp determination The expression levels of P-gp were measured by flow cytometry [37]. MCF-7/ ADR cells were treated with either fresh media or blank HPHM/TPGS2k for 12 h. The cells were washed twice with PBS, fixed with 5% paraformaldehyde for 30 min and washed with PBS again. The cells were then incubated with primary antibody (specific anti-P-gp, diluted to 1:25) for 30 min at 4  C. The unbound antibody was removed by centrifugation. After centrifugation, the cells were incubated with a secondary antibody, FITC-labeled goat anti-rabbit IgG (diluted to 1:50) for another 30 min at 4  C. The cells were kept from light exposure， washed twice with PBS and resuspended in 500 mL PBS for flow cytometer analysis. 2.12. Mitochondrial membrane potential measurement The probe 5,50 ,6,60 -tetrachloro-1,10,3,30 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1) can freely permeate cells and undergo reversible transformation from a monomer into an aggregate form when bound with high mitochondrial membrane potential. The JC-1 aggregate form is distinguished from the mono-meric form by taking on red fluorescence (590 nm) in contrast to the green fluorescence (530 nm) of momomer [38]. MCF-7/ADR cells were planted in 6-well plates at a density of 1  106 cells/well and incubated for 24 h. The blank micelles were added to the wells with fresh culture medium as a control. The cells were harvested after 2 h of incubation and stained with 500 mL of JC-1 (10 mg/mL) work solution for another 20 min. Then the cells were harvested and washed twice with of JC-1 buffer solution followed by analyzed with a multi-mode microplate reader (FilterMax F5, Molecular

Devices, USA). Results were calculated as the ratios of the cells with red fluorescence and green fluorescence intensity. 2.13. Determination of ATP content MCF-7/ADR cells were seeded in 6-well plates at a density of 1  106 cells/well and incubated for 24 h. The cells were then exposed to blank HPHM/TPGS2k for 4 h with fresh culture medium as a control. After being washed with ice-cold PBS, the cells were treated according to the operation manual of ATP assay kit. The intracellular ATP was determined by luciferin-luciferase reaction with multi-mode microplate reader (FilterMax F5, Molecular Devices, USA) in the luminescence mode. The ATP content was calculated based on the ATP standard curve and was normalized by protein concentration in each sample determined using a BCA kit. 2.14. In vivo imaging analysis Female BALB/c nude mice of 5e6 weeks age were purchased from Shanghai SLAC laboratory animal Co., Ltd. (Shanghai, China). Mice were housed at 25  C and 55% of humidity under natural light/dark conditions and were allowed free access to food and water. All animal procedures were performed in according to the protocol approved by the Animal Study Committee of Soochow University. Tumor-bearing mice were established as described previously [13]. Briefly, about 1  107 MCF-7/ADR cells were subcutaneously injected in the abdomen of the mice. Tumors were allowed to grow to approximately 50e100 mm3 before experiment. DIR-loaded HPHM and HPHM/TPGS2k were injected into the tail vein of the tumor xenograft mice. At 6 h, 12 h, 24 h and 48 h post injection, The time dependent biodistribution in MCF-7/ADR tumor-bearing nude mice was imaged by nearinfrared fluorescence imaging system (IVIS Lumina, Caliper, USA) at the time points of 6 h, 12 h, 24 h, 48 h post injection. The mice under anesthetic state via inhalation of Isoflurane were automatically moved into the imaging chamber for scanning. After 48 h, the mice were sacrificed and the major organs and tumors were harvested. Each organ or tumor was rinsed with physiological saline for three times followed by the capture of fluorescent image. 2.15. Statistical analysis All of the data are presented as the mean ± standard deviation (SD) and all experiments were performed with at least 3 independent repeats. Differences between groups were examined using Student's t-test between 2 groups or One-way analysis of variance (ANOVA) among  3 groups. A value of P < 0.05 was considered statistically significant.

3. Results and discussion 3.1. Characterization of HA-PHis and TPGS2k copolymers The chemical structure of HA-PHis and TPGS2k copolymers was confirmed by 1H NMR spectra in Fig. S1 in the Supplementary Data. The typical 1H NMR spectrum (D2O/DMSO-d6 ¼ 5:1, v/v) of HA-PHis copolymer showed peaks at da 1.30e1.39 ppm (C(CH3)2), db 2.02 ppm (COCH3), dc 7.34 ppm (NeCH]C) and dd 8.64 ppm (N]CH) (Fig. S1A). The degree of substitution (DS) of PHis, defined as the number of PHis groups per 100 sugar residues of HA copolymer, was determined by calculating the relative intensity ratio between the N-acetyl peak in HA and the methyl peaks in PHis. The calculated DS of PHis was 21.1% [39]. The typical 1H NMR spectrum (CDCl3) of TPGS2k showed peaks at da 0.86 ppm (CH(CH3)2), db 1.97e2.08 ppm (CH3 on phenyl group), dc 3.65 ppm (OCH2CH2O) and dd 3.38 ppm (OCH3) (Fig. S1B). The 1 H NMR spectra of the products indicate that HA-PHis and TPGS2k copolymers were successfully synthesized. The CMC value is an important parameter indicating the micelle forming ability of amphiphilic polymers. Fig. 2A showed the I373/ I384 fluorescence ratio of pyrene as a function of the HA-PHis concentration and the calculated CMC value was 0.0347 mg/mL Fig. 2B showed the I373/I384 fluorescence ratio of pyrene as a function of the TPGS2k concentration and the calculated CMC value of TPGS2k was 0.0186 mg/mL. The CMC value of TPGS2k was close to the CMC value of previous publication on TPGS2k by Feng SS et al. [25]. The relatively low CMC values indicate that the copolymer micelles are expected to have good dilution stability in the blood streams after intravenous injection.

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Fig. 2. CMC determination of (A) HA-PHis and (B) TPGS2k. Fluorescence intensity ratio (I373/I384) according to copolymer concentration was plotted and CMC was estimated from the threshold concentration of self-assembled micelles.

The hemolysis assay was performed to estimate the biocompatibility of the synthesized HA-PHis and TPGS2k copolymers. The level of hemolysis was compared with that of Tween 80, which is a typical surfactant approved by FDA for intravenous administration [40,41]. Hemolysis of HA-PHis, TPGS2k and Tween 80 at various concentrations are shown in Fig. S2. HA-PHis copolymer showed similar hemolysis with TPGS2k and Tween 80 at the concentration below 0.1 mg/mL，but much less hemolysis than TPGS2k and Tween 80 at the concentration above 0.3 mg/mL (P < 0.05). In addition, the HA-PHis copolymer revealed no more than 2.0% hemolysis even the concentration increased to 4 mg/mL in our previous report [29]. This indicates that the HA-PHis copolymer has much better biocompatibility than TPGS2k and Tween 80. It could be noted that as the concentration increased from 0.3 mg/mL to 0.5 mg/mL, hemolysis induced by TPGS2k and Tween 80 increased dramatically from (14.8 ± 0.40)% to (41.0 ± 2.39)% and from (8.73 ± 0.43)% to (35.7 ± 2.39)%, respectively. This indicates that the concentration of TPGS2k in the mixed micelles should be less than 0.1 mg/mL from the safety perspective. This might be that the amphiphilic polymers with polyethylene glycol block could induce the alteration of the membrane permeability at high concentration [42].

3.2. Preparation and characterization of micelles The HA-PHis and TPGS2k copolymers self-assembled into mixed micelles (HPHM/TPGS2k) in aqueous condition by a simple sonication method (Fig. 3A). In order to exclude the possibility of forming the micelle mixture composed of HA-PHis and TPGS2k mono-copolymer micelles, the co-micellization of HA-PHis and TPGS2k was characterized by dynamic light scattering (DLS). The average particle size of the binary mixed micelles was around 160 nm, which fell into the window of HA-PHis mono-micelles (190 nm) and TPGS2k mono-micelles (90 nm) (Fig. 3B). The median particle size and the relatively narrow size distribution of the obtained micelles also indicate the formation of a uniform binary mixed micelles rather than the mixture of two mono-micelles [13,43]. TEM image of HPHM/TPGS2k showed spherical and homogeneous morphology with the particle size correlated well with those obtained by DLS (Fig. 3C). As presented in Table 1, the moderate negative zeta potentials indicate that the micelles were negatively charged due to the ionization of carboxylic group of HA in the micellar shell, which might prevent the aggregation of the individual micelles through electrostatic repulsion. In addition, the

DL of DOX in HPHM/TPGS2k was 1.6-fold higher than that of HPHM, suggesting that TPGS2k played a positive role in the accommodation DOX in the micelles. 3.3. In vitro release of DOX from the micelles The in vitro release of DOX from the mixed micelles was determined under different pHs at 37  C. The copolymer micelles showed much faster DOX release at endo-lysosomal pH (~5.0) than that at physiological pH (7.4) (P < 0.05) (Fig. 3D). For instance, approximate 82.6% and 87.0% of DOX was released in 24 h from HPHM and HPHM/TPGS2k at pH 5.0 respectively in contrast to approximate 35% of DOX released at pH 7.4. The accelerated DOX release from the micelles was attributed to the physical destabilization of the hydrophobic core caused by the ionization of PHis block under acidic environment. The copolymer micelles had more compact hydrophobic micellar core composed of unprotonated PHis and VES at pH 7.4, resulting in sustained release of the incorporated DOX. When the pH decreased below 5.0, the protonated PHis blocks started to repel each other due to the same positive electrical charge, leading to the swelling of micellar core and the subsequent triggered DOX release [44e46]. It could be noted that DOX-loaded HPHM/TPGS2k displayed slightly faster drug release than DOX-loaded HPHM under the same pH condition, which was probably ascribed to the hydrophilic properties of TPGS2k improving the DOX release from the mixed micelles. The in vitro DOX release profiles indicate that the mixed micelles are expected to maintain the micellar structure integrity at physiological pH but induce rapid DOX release in the endo-lysosomes after endocytosis. 3.4. In vitro cytotoxicity studies To investigate the in vitro cytotoxicity of the HPHM/TPGS2k, the mixed micelles were tested against sensitive MCF-7 cells and P-gp overexpressing MCF-7/ADR cells [39]. No obvious growth inhibition effect was found after incubation with the blank micelles, indicating that the HA-PHis-based copolymers were nontoxic as nanocarriers (Fig. 4A). As compared to the free DOX and HPHM, the mixed micelles showed significant higher cytotoxicity against MCF7/ADR cells (P < 0.05), suggesting that TPGS2k in the mixed micelles accounted for the enhanced cytotoxicity. The RI and RF were calculated to quantitatively access the reversal effect on MDR by the mixed micelles. As shown in Table 2,

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Fig. 3. Characterization of mixed micelles (HPHM/TPGS2k). (A) Schematic illustration of HPHM/TPGS2k comprised of HA-PHis and TPGS2k copolymers. (B) Particle size distribution of HPHM/TPGS2k. (C) Transmission electron microscopy (TEM) image of HPHM/TPGS2k. Scale bar represents 200 nm. (D) pH-sensitive release profile of DOX from HPHM and HPHM/TPGS2k in PBS (pH 7.4 and 5.0) at 37  C. Data as mean values ± SD (n ¼ 3).

the RI value of DOX was 45.45, suggesting the good resistance of MCF-7/ADR cells to DOX. The RI value of HPHM and HPHM/TPGS2k remarkably decreased to 8.10 and 1.55 respectively. The partial reversal effect revealed by HPHM was attributed to the synergistic effect of uptake way of micelles evading the P-gp efflux pump and the pH triggered DOX release, which increased the concentration of DOX in the MCF-7/ADR cells. The HPHM/TPGS2k showed much higher RF values than the other groups, demonstrating the best reversal effect on MDR cells due to the presence of TPGS2k. Furthermore, HPHM/TPGS and HPHM/TPGS presented the same cytotoxicity and MDR reversal effect in MCF-7/ADR cells, suggesting that overcoming MDR effect of TPGS2k is similar to TPGS in the mixed micelles.

3.5. Intracellular accumulation of DOX To further evaluate the effect of the TPGS2k on the P-gp mediated drug efflux, intracellular accumulation of DOX in MCF-7 and MCF-7/ADR cells were measured using flow cytometer respectively. The cellular uptake of DOX after incubation with different formulations for 2 h was presented in Fig. 4B, C and D. There was no significant difference in DOX accumulation in MCF-7 cells from all the HA-PHis-based copolymer micelles (P > 0.05). This suggests that the recognition of CD44 receptors and pH-sensitive micellar core of the mixed micelles were not influenced by the TPGS2k. The accumulation of free DOX in MCF-7 cells was 6.6-fold higher than that in MCF-7/ADR resistant cells (P < 0.05), indicating that P-gp

Table 1 Characterization of DOX-loaded HA-PHis-based micelles. Copolymer micelles

Mean diameter (nm)

Polydispersity

Zeta potential (mV)

EEa (%)

LCb (%)

HPHM HPHM/TPGS HPHM/TPGS2k

187.8 ± 8.1 172.2 ± 9.7 154.1 ± 5.3

0.083 ± 0.041 0.110 ± 0.037 0.037 ± 0.033

11.6 ± 0.47 12.2 ± 0.91 13.4 ± 0.77

89.0 ± 2.11 93.0 ± 1.57 92.2 ± 2.49

6.29 ± 0.47 10.06 ± 1.05 9.93 ± 0.16

a b

EE (%) ¼ encapsulation efficiency. LC (%) ¼ drug loading content.

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Fig. 4. (A) Viability of MCF-7 cells and MCF-7/ADR cells after treatment with blank micelles at different concentrations for 48 h. (B) Flow cytometry measurement of the intracellular uptake of DOX in the MCF-7 cells and MCF-7/ADR cells treated with different DOX-loaded copolymer micelles or free DOX. (C) Flow cytometry histograms of DOX accumulation in the MCF-7 cells and (D) MCF-7/ADR cells at 2 h. Data as mean values ± SD (n ¼ 3).

overexpression impeded DOX accumulation in the drug resistant cells. The HPHM showed 7.0-fold higher uptake efficiency of DOX in MCF-7/ADR cells than free DOX (P < 0.05). This was attributed to the synergistic effect of receptor-mediated endocytosis of HPHM bypassing P-gp mediated efflux and the pH-triggered drug release as well as the PHis facilitated endo-lysosomal escape. Furthermore, the mixed HPHM/TPGS2k showed 12.7-fold higher uptake efficiency of DOX than free DOX (P < 0.05) and 1.8-fold higher than HPHM (p < 0.05). This indicates that the combination of enhancement of drug accumulation in MDR cells and inhibition of P-gp mediated efflux can serve as an efficient way to reverse tumor MDR. In addition, no significant difference in DOX uptake from the HPHM/TPGS and HPHM/TPGS2k in MCF-7/ADR cells (P > 0.05), suggesting that the TPGS2k was similar to TPGS in terms of MDR reversal. 3.6. Intracellular pH-triggered release of DOX Chloroquine, a weak base that could increase the pH the acid organelles was used to evaluate whether acid endo-lysosome condition could indeed trigger the DOX release from the micelles. Fig. 5 shows the cellular distribution of DOX released from HPHM/TPGS2k with or without pre-incubation of chloroquine in the MCF-7/ADR cells. The red fluorescence of DOX was similar in the absence and presence of chloroquine at early time points, indicating that the uptake of HPHM/TPGS2k was not affected by

chloroquine. The cells without treating with chloroquine showed DOX distribution in the cytoplasm after incubation for 0.5 h, as characterized by a co-localization of red fluorescence with green fluorescence of endo-lysosomes that producing a yellow fluorescence (white arrow) (Fig. 5A). The red fluorescence of DOX was increased in the cytoplasm following 2 h incubation. After a prolonged incubation (4 h), it was noted that strong DOX fluorescence was observed in the cytoplasm while the yellow fluorescence was gradually disappeared. This phenomenon was probably attributed to the protonation of PHis in the acidic organelles which facilitated the endo-lysosomal escape of DOX and subsequent distribution in the cytoplasm. However, the cells treated with chloroquine revealed DOX localization in endo-lysosomes, as characterized by the yellow fluorescence (white arrows) at different times (Fig. 5B). The presence of chloroquine could increase the pH of the endo-lysosome compartments, which inhibiting the protonation of PHis block, leading to low DOX release from the micelles and trapping in endo-lysosomes. The results indicate that the HPHM/TPGS2k rapidly disassembled in the acidic endo-lysosomes, inducing the accelerated DOX release and distribution into the cytoplasm via PHis facilitated endolysosomal escape. Furthermore, it could be assumed that TPGS2k released from the disassembled mixed micelles was also transported into the cytoplasm, which inhibited the P-gp efflux and increased the intracellular DOX accumulation in MCF-7/ADR cells.

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Table 2 The IC50 and MDR reversal effect on the MCF-7 and MCF-7/ADR cells. Formulations

IC50 MCF-7

DOX HPHM HPHM/TPGS HPHM/TPGS2k a b

2.40 1.74 1.82 1.79

± ± ± ±

RIa

RFb

45.45 8.10 1.54 1.55

1.00 7.74 38.54 39.52

MCF-7/ADR 0.08 0.13 0.11 0.09

109.08 14.09 2.83 2.76

± ± ± ±

5.34 0.41 0.18 0.18

RI ¼ resistant index. RF ¼ reversal factor.

3.7. Mechanisms of the MDR reversal effect of mixed micelles The fluorescent dye Rh123 is a substrate of P-gp, which is commonly used for evaluation of P-gp activity in resistant cancer cells [47]. In this study, the effect of HA-PHis-based copolymer micelles on Rh123 accumulation in MCF-7/ADR cells was measured. As shown in Fig. 6A, the MCF-7/ADR cells incubated with either HPHM/TPGS2k or HPHM/TPGS showed higher Rh123 accumulation than those incubated with HPHM and Rh123 solution (P < 0.05), suggesting that TPGS2k in the mixed micelles played a positive role in inhibition of P-gp activity. The MCF-7/ADR cells showed similar Rh123 accumulation after incubation with HPHM and Rh123 solution (P > 0.05), indicating that the moderate MDR reversal effect caused by HPHM was attributed to the above mentioned enhanced accumulation of DOX in the cells rather than the inhibition of P-gp activity. In addition, similar results were observed in Rh123 efflux test (Fig. 6B). The MCF-7/ADR cells exhibited similar Rh123 efflux after incubation with HPHM or Rh123 solution (P > 0.05), but higher Rh123 efflux than those incubated with HPHM/TPGS2k or HPHM/TPGS (P < 0.05). These results suggest that enhancement of the accumulation of cytotoxic drug in the MDR cells by receptormediated endocytosis and endo-lysosomal escape can only achieve limited MDR reversal effect. The combination of receptormediated endocytosis, endo-lysosomal escape and inhibition of Pgp activity offers a more efficient approach to reverse tumor MDR. To further evaluate whether the mixed micelles directly influence the P-gp levels, P-gp expression of the MCF-7/ADR cells was detected by flow cytometry. Fig. 6C shows the effect of HPHM/

TPGS2k on P-gp-specific antibody binding. No significant difference was observed between HPHM/TPGS2k and the control group (P > 0.05). Therefore, HPHM/TPGS2k could not suppress the P-gp expression of the MCF-7/ADR cells. Moreover, the effect of the HPHM/TPGS2k on cellular energy metabolism of the MCF-7/ADR cells was examined. The change in mitochondrial membrane potential was first measured using a lipophilic membrane potentialsensitive dye JC-1 [35]. Mitochondrial depolarization is indicated by a decrease in the ratio of red/green fluorescence intensity. As shown in Fig. 7A, the values of JC-1 red/green significantly decreased after incubation with HPHM/TPGS2k in the MCF-7/ADR cells as compared to the control group (P < 0.05). Furthermore, the ATP levels of the MCF-7/ADR cells after treatment with HPHM/ TPGS2k are shown in Fig. 7B. HPHM/TPGS2k induced a significant decrease in the intracellular ATP levels as compared to the control group (P < 0.05). Because P-gp is an ATP-dependent multidrug efflux pump, which is involved in energy metabolism linking with mitochondria membrane [38,48]. A high mitochondria membrane potential is required to maintain the normal activity of the ATP metabolism [49]. Therefore, the reduction of membrane potential and ATP level caused by HPHM/TPGS2k would result in the inhibition of P-gp mediated drug efflux and the enhancement of the drug accumulation in the drug-resistance cells. All these results indicate that the synergistic effect of the enhancement of DOX accumulation in MCF-7/ADR cells arising from receptor mediated endocytosis, pH triggered DOX release, PHis facilitated endolysosomal escape and the inhibition of P-gp mediated DOX efflux account for MDR reversal.

3.8. In vivo tumor-targeting observed by NIRF imaging The in vivo biodistribution and tumor-targeting efficiency of the copolymer micelles were evaluated using a non-invasive near infrared optical imaging technique. The tumor-bearing mice were injected with DIR-loaded HPHM and HPHM/TPGS2k, respectively. As shown in Fig. 8A, the real-time images of micelles in the live mice were monitored at 6 h, 12 h, 24 h and 48 h after administration. Most of the DIR accumulated in liver and tumor after administration of both micelles. With the prolongation of time, the

Fig. 5. The confocal microscope images of MCF-7/ADR cells incubated with HPHM/TPGS2k (A) in the absence of chloroquine and (B) in the presence of chloroquine for 0.5 h, 2 h and 4 h. Blue, green and red colors indicate Hoechst 33342, LysoTracker green and DOX, respectively. Scale bars correspond to 10 mm in all the images. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. The effect of the blank mixed micelles on P-gp efflux activity in the MCF-7/ADR cells. (A) The effect of the mixed micelles on Rh123 accumulation and (B) Rh123 efflux. (C) The effect of the mixed micelles on P-gp expression in the MCF-7/ADR cells. Data as mean values ± SD (n ¼ 3).

fluorescence intensity in the tumor region was gradually increased. At 12 h post injection, the maximum fluorescence was observed and the fluorescence could last for more than 48 h in the tumor region. The tumor targeting of the micelles could be attributed to a combination of EPR effect and receptor-mediated uptake of micelles [50]. In particular, the fluorescence intensity of the HPHM/ TPGS2k in the tumor region was stronger than that of the HPHM during the monitoring period. This suggests that PEGylation is capable of increasing the circulation time of micelles in the bloodstream, resulting in the accumulation at the tumor site via

EPR effect [51]. However, it was noteworthy that both of the micelles showed strong fluorescence intensity in the liver at all the times. The mainly accumulation in the liver might be caused by reticuloendothelial system (RES) [52] and a HA receptor in the liver sinusoidal endothelial cells for endocytosis [53,54]. Therefore, further chemical modifications of HA is needed to reduce the accumulation in the normal organs in the future. The major organs and tumor tissues were isolated for further study. The HPHM/TPGS2k showed stronger fluorescence intensity in tumor tissue than HPHM (Fig. 8B). In the quantitative analyses,

Fig. 7. The effect of the blank mixed micelles on (A) mitochondrial membrane potential and (B) intracellular ATP in the MCF-7/ADR cells. Data as mean values ± SD (n ¼ 3).

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Fig. 8. (A) In vivo imaging of tumor-bearing mice administrated with DIR-loaded micelles. Images taken after administration of HPHM at 4 h (a1) and 12 h (a3), and HPHM/TPGS2k for 4 h (a2) and 12 h (a4), respectively. (B) Ex vivo fluorescence images of tumors and organs collected at 12 h post-injection of HPH/TPGS2k (b1) and HA-PHis micelles (b2). (C) Quantification of the ex vivo tumor uptake characteristics of micelles. Uptake expressed as fluorescence per mm2 of tumor. Data as mean values ± SD (n ¼ 3).

the mixed micelles exhibited more than 1.37-fold higher intensity in the tumor tissue than HPHM (P < 0.05) (Fig. 8C). The prolonged circulation time of HPHM/TPGS2k is thought to be related to the serum stability offered the PEG shell. These results indicate that the mixed HPHM/TPGS2k can be served as a highly efficient carrier to overcome MDR. 4. Conclusion In this study, a pH-sensitive mixed micelles composed of HAPHis and TPGS2k copolymers was developed to overcome MDR. HA-PHis increased the intracellular uptake via CD44 receptormediated endocytosis, pH-triggered drug release and PHis facilitated endo-lysosomal escape. TPGS2k in the mixed micelles was found to further enhance the drug accumulation in the MCF-7/ADR cells by inhibition of P-gp mediated drug efflux because of the HPHM/TPGS2k reduction of mitochondrial membrane potential and ATP level in the cells. The copolymer micelles have been demonstrated to be a potential nanocarrier to overcome tumor MDR. Acknowledgments This work was supported by National Natural Science Foundation of China (No. 81173004, 81302713, No. 81202473 and No. 81302719), Natural Science Foundation of Jiangsu Province (BK2012182), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Appendix A. Supplementary data

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Please cite this article in press as: Qiu L, et al., Enhanced effect of pH-sensitive mixed copolymer micelles for overcoming multidrug resistance of doxorubicin, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.08.008