Active-targeting docetaxel-loaded mixed micelles for enhancing antitumor efficacy

Journal of Molecular Liquids 264 (2018) 172–178

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Active-targeting docetaxel-loaded mixed micelles for enhancing antitumor efficacy Chunhuan Shi a,b, Zhiqing Zhang c, Fang Wang c, Yuxia Luan a,⁎ a b c

School of Pharmaceutical Science, Shandong University, 44 West Wenhua Road, Jinan, Shandong Province 250012, PR China Department of Pharmacy, Dongying People's Hospital, 317 Nanyi Road, Dongying, Shandong Province 257091, PR China College of Science, China University of Petroleum, 66 West Changjiang Road, Qingdao, Shandong 266580, PR China

a r t i c l e

i n f o

Article history: Received 9 April 2018 Received in revised form 6 May 2018 Accepted 8 May 2018 Available online 09 May 2018 Keywords: Docetaxel Folic acid Micelles Active-targeting

a b s t r a c t Active targeting agents can identify patients with receptors overexpressed on the surface of cancer cells. The selective and efficient drug delivery to tumor cells can remarkably improve different cancer therapeutic effect. Folic acid (FA) conjugation is a facile approach for targeting folate receptor-expressing tissues for personalized treatment. In the present study, ethoxy poly(ethylene glycol)- folic acid (FA-PEG) is introduced as the potent targeting moieties in docetaxel (DTX)-loaded micelles. The spherical FA-PEG/PEO–PPO–PCL mixed micelles revealed a narrow distributed size at 106.1 ± 0.3 nm. The low critical micelle concentration (CMC) of the mixed micelles (about 3.8 μg mL−1) indicated the excellent self-assembly ability in water and predominant stability against dilution in the circulation. The in vitro drug release of DTX in micelles presented a controlled and sustained release pattern. Moreover, in vitro cytotoxicity results showed the FA-PEG/PEO–PPO–PCL micelles exerted higher cytotoxicity compared with PEO–PPO–PCL micelles on FR-positive MCF-7 cells. Cellular uptake studies clearly demonstrated that FA-PEG micelles were more efficiently accumulated in MCF-7 cells via FRs mediated endocytosis. Therefore, the prepared FA-PEG/PEO–PPO–PCL micelles can provide a promising tumor-targeting drug delivery system for efficient cancer therapy. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Cancer is one of the major causes of death worldwide. In cancer chemotherapy, the use of anticancer drugs has been limited by their toxic side effects in normal organs and an inadequate concentration of the drug at tumor sites [1]. Docetaxel (DTX) is an important anticancer taxane, which exhibits therapeutic activity against a broad spectrum of tumors, such as breast cancer, prostate cancer, non-small cell lung cancer for decades [2,3]. Despite the favorable antitumor effects, several limitations including poor aqueous solubility, serious untoward toxicity, as well as drug resistance have restricted the clinical use of DTX [4]. In contrast, targeted agents can identify patients with receptors overexpressed on the surface of cancer cells, thus allowing drug accumulation in tumor tissue and achieving treatment with a desirable targeted therapeutic [5–7]. Therefore, development of tumor-targeted delivery systems of DTX is one of the urgent needs in drug delivery research. To date, many investigators have employed natural endocytosis pathways for targeting macromolecules or particulate carriers into cells. To achieve enhanced targeting efficiency, various moieties e.g. ⁎ Corresponding author. E-mail address: [email protected] (Y. Luan).

https://doi.org/10.1016/j.molliq.2018.05.039 0167-7322/© 2018 Elsevier B.V. All rights reserved.

antibodies, proteins, peptides and small molecules were decorated with drug nanocarriers to examine their specific interactions with tumor cells. Conventional cancer treatment has been greatly improved by the addition of an increasing number of new molecularly targeted agents. Folic acid (FA) is a kind of vitamin with low toxicity and ready availability, could bind folate receptor (FR) with extremely high affinity [8–10]. Moreover, FA is one of the essential ingredients of cell metabolism, growth, proliferation, survival of cells as well as synthesis, repair of DNA [11,12]. There are many evident features that make FA attractive for drug delivery including low immunogenicity, high binding affinity with FR, ease of conjugation, stability during storage, more tumor selectivity and ready availability [13,14]. It is well known that the folate receptor (FR), a kind of folate-binding protein, which can bind folate and folate conjugates with extremely high affinity, is a confirmed target for tumor-specific drug delivery [15–17]. Importantly, FR is commonly at low levels in normal tissues, but vastly overexpressed in a wide variety of human tumors including the human breast cancer cell lines of MCF-7 [18,19]. Accordingly, FA is widely applied in drug delivery systems to facilitate the efficiency of cellular uptake and the therapeutic effects. Polymeric micelles have sparked considerable interest because of their technical simplicity and prominent superiorities, such as the high stability [20], prolonged systemic circulation [21], ease of active target

C. Shi et al. / Journal of Molecular Liquids 264 (2018) 172–178

modification [22], the great flexibility in tuning drug solubility [23] and so on. Based on our previous synthesized amphiphilic triblock copolymers poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(e-caprolactone) (PEO–PPO–PCL) [24,25] and active targeting methoxy poly(ethylene glycol)- folic acid (FA-PEG), the DTX-loaded FA-PEG/ PEO-PPO-PCL mixed micelles were constructed for the enhanced target specificity and improved anti-cancer efficiency of DTX. PEO–PPO–PCL and FA-PEG copolymers could self-assemble into mixed micelles at low critical micelle concentration (CMC) in aqueous solutions. The entrapment efficiency, drug loading, morphology, particle size, zeta potential and CMC were characterized. Moreover, the drug delivery system was evaluated with the in vitro release behavior, FRs mediated cellular uptake and cytotoxicity against FRs-positive MCF-7 and FRs-negative A549 cells. The results demonstrate that the constructed mixed micelles have shown good sustained drug release behavior, excellent cellular uptake efficiency and predominant anticancer potency against FRspositive cancer cells. Thus, the prepared mixed micelles provide a promising platform for enhancing the therapeutic efficacy of DTX.

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The amount of DTX encapsulated in the mixed micelles was measured by high performance liquid chromatography (HPLC, Agilent LC1100). The solution was then filtered using a 0.22 μm syringe and examined by HPLC. 20 μL sample solution was injected in triplicate using a Hypersil-ODS2 column (4.6 mm × 250 mm, 5 μm) (Elite, China). The mobile phase was a mixture of acetonitrile and water (65: 35, v/v) and the flow rate of the mobile phase was 1.0 mL min−1. The column effluent was detected at 230 nm using a UV–Vis detector. Drug loading (DL%) and encapsulation efficiency (EE%) were calculated by the following Eqs. (1) and (2): DLð%Þ ¼

Weight of drug in micelles  100% Weight of micelles

ð1Þ

EEð%Þ ¼

Weight of drug in micelles  100% Weight of feeding drug

ð2Þ

2.4. CMC determination of the mixed micelles 2. Materials and methods 2.1. Materials Docetaxel of 99.8% purity was purchased from Dalian Meilun Biotech Co., Ltd. FA-PEG (MW 2000) was purchased from Shanghai Ponsure Biotech. Inc. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), coumarin-6 and dimethylsulphoxide (DMSO) were purchased from Sigma-Aldrich (China). Cellulose ester membranes (dialysis bag) with a molecular weight cut-off value (MWCO) of 3500 (Greenbird Inc., Shanghai, China) were used in the dialysis study. PEO–PPO–PCL was synthesized in our lab [20]. Folate-free RPMI-1640 medium, fetal bovine serum (FBS), 0.25% (w/v) trypsin–0.03% (w/v) EDTA, plastic cell culture dishes and plates and phosphate buffer solution (PBS) were purchased from Gibco BRL (Gaithersberg, MD, USA). Water used in the experiment was doubly distilled. The acetonitrile (Shanghai Siyou Co., Ltd., China) was HPLC grade. All other solvents were analytical- or chromatographic-grade. 2.2. Preparation of mixed micelles DTX-loaded FA-PEG/PEO-PPO-PCL micelles were prepared by thin film hydration method. DTX (1.0 mg), PEO–PPO–PCL (20.0 mg) and FA-PEG (5.0 mg) were co-dissolved in acetonitrile and then the solution was removed by rotary vacuum evaporation. The film formed was additionally hydrated with 1 mL water, incubated at 60 °C for 1 h. Finally, the resultant mixture was filtered through 0.22 μm polyethersulfone syringe filter in a sterile and then micelles were successfully prepared. 2.3. Characterization of DTX-loaded mixed micelles The particle size, polydispersity and zeta potential of mixed micelles were measured by dynamic light scattering (DLS, BIC·Brook-Haven). All samples were performed in triplicate and the measurement was carried out at 25 °C with a laser light of wavelength 532 nm and 90° scattering angle. The transmission electron microscope (TEM, JEM-200CX) was utilized to observe the morphology of mixed micelles. A drop of mixed micelles was placed onto 300-mesh copper grids coated with carbon without staining. Then, the copper grid was dried for about 30 min and then observed by TEM.

The critical micelle concentration (CMC) was determined by the fluorescence technique using pyrene as a fluorescence probe performedon F-7000 fluorescence spectrofluorophotometer. Pyrene, which was dissolved in acetone, was dispersed in mixed micelles solutions with concentration ranging from 0.2 to 30 mg L−1. The final concentration of pyrene was 6 × 10−7 M in the solution. The solutions were equilibrated at room temperature in dark overnight after 30 min of ultrasonication. The emission spectrum of these solutions from 300 to 450 nm (bandwidth = 2.5 nm) was recorded by fluorescence spectrophotometer (Hitachi F-7000, Japan) with the excitation wavelength fixed at 334 nm. The CMC value of the mixed micelles was evaluated from the plot of the intensity ratio of the first peak (I1, 373 nm) and the third peak (I3, 384 nm) versus polymer concentration. 2.5. In vitro drug release studies To verify the release properties of DTX from mixed micelles, in vitro release experiments were conducted using dialysis method. Samples were placed in a dialysis bag (molecular weight cut-off 3500 Da). The dialysis bags were immersed in phosphate buffer saline (PBS, 0.15 M, pH 7.4) containing 0.5% (w/v) Tween 80, and then were gently shaken in a water bath at a constant temperature (37 °C). DTX release from stock solution was also conducted under the same condition as a control. At the defined time intervals, 1.0 mL medium was withdrawn and the medium was refreshed with an equal volume of the same buffer. To estimate the amount of DTX release, the drug in the release medium at each sampling point was measured by HPLC at 230 nm. The results of triplicate measurements were used to calculate cumulative drug release. 2.6. Hemolytic evaluation The hemolytic potential of the drug-loaded micelles was investigated using rabbit red blood cells (RBC). Briefly, 1.0 mL RBC suspension (2% w/v) was dispersed in 4.0 mL distilled water and 4.0 mL normal saline as positive control and negative control, respectively. 1.0 mL micelles solutions with different concentrations was added to 3.0 mL of normal saline and interacted with 1.0 mL RBC suspension. All of the samples were incubated in a 37 °C water bath for 2 h and then centrifuged at 3000 rpm for 10 min. The absorbance of the supernatant at

Table 1 The drug loading (DL), encapsulation efficiency (EE), zeta potential and size of mixed micelles (mean ± SD, n = 3). Formulation

DL%

EE%

Zeta potential

Size

PDI

Mixed micelles

4.21 ± 0.02

91.33 ± 0.42

−0.27 ± 0.11

106.1 ± 0.3

0.14 ± 0.02

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Fig. 1. The size (A) and morphology (B) of DTX-loaded FA-PEG/PEO-PPO-PCL mixed micelles determined by DLS and TEM.

540 nm was measured by an ultraviolet spectrophotometer and the hemolysis was calculated using formula (3).

Hemolysisð%Þ ¼

Asample −Anegative  100%: Apositive −Anegative

ð3Þ

2.7. In vitro cell experiments 2.7.1. Cell lines and cell culture In order to determine the anticancer effect of the mixed micelles, the samples were evaluated against two kinds of cell lines including FRs-positive human breast cancer cells (MCF-7) and FRs-negative human lung carcinoma cells (A549). MCF-7 and A549 cells were kindly donated by the Department of Pharmacology, School of Pharmacy, Shandong University. Cells were cultured in folate-free RPMI medium, supplemented with 10% fetal bovine medium (FBS) and were incubated in a humidified 5% CO2 incubator at 37 °C. The medium was changed every other day. The cells were subcultured 2–3 times per week with 0.25% trypsin–EDTA. Before measurements, the cells were cultured until confluence was reached to about 75%.

Fig. 2. The I373/I384 ratio of fluorescence emission spectra of pyrene versus concentration of FA-PEG/PEO-PPO-PCL micelles.

2.7.2. The cellular uptake studies and flow cytometric (FCM) analysis The hydrophobic fluorescent dye coumarin-6 (C6) was used as a probe to substitute DTX. C6 was incorporated into the formulations as the same procedure described in Section 2.2. The FRs positive MCF-7 and the FRs negative A549 cells were seeded in a 6-well plate with a density of 2 × 105 cells/well, which were cultured for 24 h at 37 °C. The cells were cultured with C6-loaded PEO-PPO-PCL micelles (folatefree medium), C6-loaded FA-PEG/PEO-PPO-PCL micelles (folate-free medium) and C6-loaded FA-PEG/PEO-PPO-PCL micelles (folate medium) respectively for 2 h. The cells were washed with PBS for three times and harvested via trypsinization, which was followed by collecting via centrifugation (1000 rpm × 5 min) and resuspending in PBS. Finally, flow cytometry was utilized to quantify the fluorescence intensity. 2.7.3. Cytotoxicity assay in vitro The in vitro cytotoxicity of blank FA-PEG/PEO-PPO-PCL mixed micelles, free DTX, and DTX-loaded FA-PEG/PEO-PPO-PCL mixed micelles against two different cancer cell lines including MCF-7 and A549 cells, were determined by the MTT dye reduction assay. Briefly, MCF-7 and A549 cells in the logarithmic growth phase were seeded at a density of 5000 cells well in 96-well plates. Following by attachment overnight, samples were diluted with the culture medium to prepare various concentrations of blank micelles, free DTX, and DTX-loaded FA-PEG/PEO-

Fig. 3. In vitro release curves of DTX from the mixed micelles (mean ± SD, n = 3).

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3. Results and discussion 3.1. Characterization of mixed micelles

Fig. 4. The hemolysis ratio (%) of the DTX-loaded micelles at different concentrations (mean ± SD, n = 3).

PPO-PCL, and tested in triplicate (200 mL each). At scheduled time intervals (24 h and 48 h), 10 μL of 5 mg mL−1 MTT dissolved in PBS was added to each well and the plates were incubated for 4 h at 37 °C. After removing the medium, 150 μL of DMSO was added to each well to dissolve the formed purple crystals derived from MTT and the plate was vigorously shaken before measurement. The absorbance of each well was read on a microplate reader (Enspire instruments, Perkin Elmer, America) at a wavelength of 570 nm. Results were presented as cell inhibitory rate and half maximal inhibitory concentration (IC50) values. The percentage of cell growth inhibition was calculated as follows: cell inhibitory rate = (A570control − A570sample) / A570control × 100%, where untreated cells are tested as controls. The cytotoxicity of micelles was expressed as IC50 values, which were calculated by using nonlinear regression analysis.

2.8. Statistical analysis Data are expressed as mean ± standard deviation (SD) from at least three separate experiments.

DTX-loaded FA-PEG/PEO-PPO-PCL micelles were prepared by thin film hydration method. Drug loading (DL), encapsulation efficiency (EE), size with polydispersity (PDI), zeta potentials are presented in Table 1. The DL and EE of the mixed micelles is (4.21 ± 0.02)% and (91.33 ± 0.42)%, respectively. The micelles size and size distribution could play an important role in determining in vitro and in vivo drug release kinetics, cellular uptake and biodistribution of the micelles [26]. In order to achieve longevity during systemic circulation, micelles must be small enough to evade detection and destruction by the reticuloendothelial system (RES). As illustrated in Table 1, the mean particle size of the mixed micelles is approximately (106.1 ± 0.3) nm with comparatively low PDI of 0.14 ± 0.02. Particles in this size range can escaping from the phagocytosis of the reticuloendothelial system and be selectively taken up by tumors because of the higher vascular permeability of the tumor cells compared to normal tissue [27,28]. Hence, the size of micelles on this study is suitable for tumor-specific accumulation via the enhanced permeability and retention (EPR). The zeta potential of the mixed micelles is slightly negative, about (−0.27 ± 0.11) mV. The negatively charged micelles can avoid the strong forces with serum proteins and macrophage uptake in the circulation system [29–31]. It can be seen from Fig. 1 that the micelles were relatively uniform in size and spherical in shape.

3.2. CMC determination of the mixed micelles CMC is an important parameter for indicating the stability of micelles, both in vitro and in vivo. In the process of micelles formation, the hydrophobic pyrene can transfer from the aqueous phase into the hydrophobic core, which brought about an abrupt increase of its fluorescence intensity, resulting in the change of intensity ratios of I373/I384 around the CMC [32]. As shown in Fig. 2, it could be seen that the I373/I384 ratio decreases with increasing the concentration of polymers. With an increase in concentration of FA-PEG/PEOPPO-PCL, an abrupt decrease of intensity ratio of I 373/I 384 was observed, which indicated the formation of the mixed micelles. The I373/I384 ratio decreases dramatically around 3.8 μg mL −1 , defined as the CMC. The low value of CMC was important for drug delivery, as the low CMC can make the drug delivery system intact during their circulation in blood. Our mixed micelles with low CMC value indicate they are insensitive to dilution and can keep a longer circulation time in vivo.

Fig. 5. Fluorescence intensity of MCF-7 cells and A549 cells analyzed by flow cytometric. Control (black), C6-loaded FA-PEG/PEO-PPO-PCL (red), C6-loaded PEO-PPO-PCL (blue), C6-loaded FA-PEG/PEO-PPO-PCL in folate medium (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Cytotoxicity of blank micelles on MCF-7 cells and A549 cells.

3.3. In vitro drug release studies To serve as good drug carriers and promote drug extravastation into tumors via the EPR effect, it is crucial for micelles to be able to entrap the drug for an extended period of time during circulation. The in vitro

release profile of DTX is shown in Fig. 3. It can be observed that the free DTX show an almost complete drug release 91% after 12 h. However, DTX released from the mixed micelles were much slower in the process, which occurred in a controlled and sustained manner. Approximately 40% of DTX was released from the dialysis after the same time

Fig. 7. Cytotoxicity study of different samples in MCF-7 cells and A549 cells after 24 h and 48 h incubation. (A) free DTX, (B) DTX-loaded PEO-PPO-PCL micelles, (C) DTX-loaded FA-PEG/ PEO-PPO-PCL micelles. (*: P b 0.05, comparing DTX-loaded FA-PEG/PEO-PPO-PCL micelles with DTX-loaded PEO-PPO-PCL micelles).

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(12 h). Free DTX molecules could quickly penetrate through the dialysis bags and diffuse into the release medium. Nevertheless, encapsulated DTX firstly need to be released from the carrier and then diffuse out the dialysis membrane, which results in the lower drug release rate than free DTX sample.

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rate of the formulations increased with an increasing DTX concentration from 0.025 μg mL−1 to 25 μg mL−1. Especially, the cell inhibition capacity was significantly enhanced for MCF-7 cells incubated with FA-PEG/ PEO-PPO-PCL micelles in comparison to PEO-PPO-PCL micelles, demonstrating the excellent killing ability was improved by the FA-PEG component for MCF-7 cells.

3.4. Hemolytic evaluation 3.7. IC50 values of DTX-loaded mixed micelles Since the mixed micelles are designed to be administrated via intravenous injection, it is important to illustrate in vitro hemolytic activity, which can also furnish the additional information in regard to the biocompatibility of the mixed micelles in the case of the in vivo application. The hemolysis ratio of the micelles is presented in Fig. 4. The mixed micelles showed inappreciable hemolytic potential (b5%) to RBCs even at the highest concentration of 40 mg mL−1, indicating they have excellent blood compatibility. The low hemolysis ratio can be attributed to the protective layer of PEO in micelles [33–35]. 3.5. The cellular uptake studies and flow cytometric (FCM) analysis To investigate folate-assisted endocytosis, a quantitative folate targeting cellular uptake test is carried out using coumarin-6 (C6) loaded micelles on FRs-negative A549 cells and FRs-positive MCF-7 cells. In our previous study, it has been demonstrated that the uptake efficiency of PEO-PPO-PCL micelles was higher than that of free drug [25]. In the present study, a competitive binding study of the FRs was investigated to identify that the enhanced cellular uptake was directly associated with FRs mediated endocytosis. The results of cellular uptake were quantitatively confirmed by flow cytometry. As illustrated in Fig. 5, for A549 cells [FRs(−)], FA-PEG/PEO-PPO-PCL micelles had similar accumulation in as compared to those of PEO-PPO-PCL micelles. In contrast, in the case of MCF-7 cells [FRs(+)], after 2 h incubation at 37 °C, the cellular uptake of FA-PEG/PEO-PPO-PCL micelles were much greater than that of PEO-PPO-PCL micelles. This improved uptake is ascribed to the specific binding between FA in the mixed micelles and FRs on the surface of MCF-7 cells. However, when MCF-7 cells were incubated with FA-PEG/PEO-PPO-PCL micelles, much greater cellular uptake was observed in the folate-free medium than in the folate medium. The results indicated that free folic acid added in the culture medium prevented FAPEG/PEO-PPO-PCL micelles from transporting into MCF-7 cells by competitive binding to folate receptors on the cell surface. These results clearly demonstrate that the superiority of FA-PEG/PEO-PPO-PCL micelles to increase the accumulation of drug in tumors overexpressed folate receptors. 3.6. In vitro cytotoxicity of blank micelles and DTX-loaded mixed micelles In vitro cytotoxicity of blank micelles was evaluated by MTT assays. As revealed in Fig. 6, in the concentration ranges (0.5–500 μg mL−1) used in this study, the viability of MCF-7 and A549 cells after incubation with blank micelles is maintained above 85% at all tested concentrations after 24 h and 48 h incubation. Therefore, it confirmed the blank micelles are expected to be safe for their biomedical applications. To evaluate the cytotoxicity of the DTX-loaded micelles, in vitro cytotoxicity tests of DTX-loaded micelles against MCF-7 and A549 cell lines were also carried out. As revealed in Fig. 7, a dose-dependent cytotoxicity was observed. For both MCF-7 and A549 cells, the inhibition Table 2 The IC50 of different samples on MCF-7cells and A549 cells. Samples

DTX DTX loaded PEO-PPO-PCL micelles DTX loaded FA-PEG/PEO-PPO-PCL micelles

IC50 (μg mL−1) MCF-7 cells

A549 cells

25.47 ± 0.90 15.47 ± 1.32 6.75 ± 1.50

30.43 ± 1.16 16.73 ± 1.21 18.36 ± 0.99

The efficacies of killing tumor cells by different formulations are further quantitatively demonstrated by the IC50 value (drug concentrations that kill 50% of cells). As displayed in Table 2, compared with free DTX and DTX-loaded PEO-PPO-PCL micelles, DTX-loaded FA-PEG/PEO-PPOPCL micelles exhibited the most excellent antitumor effects on MCF-7 cells, with the lowest IC50 values (6.75 ± 1.50) μg mL−1. Especially, relatively greater antitumor efficacy were observed in MCF-7 cells [FRs (+)] treated with DTX-loaded FA-PEG/PEO-PPO-PCL in contrast that with A549 cells [FRs(−)], about 3-fold more effective. Therefore, it could be inferred that the cytotoxicity is closely related to the FAreceptor on the cancer cells. 4. Conclusions In summary, the active-targeting DTX-loaded FA-PEG/PEO-PPO-PCL micelles were successfully prepared for efficient anticancer therapy. The self-assembled micelles had the desirable size (106.1 ± 0.3 nm), low CMC value (3.08 μg mL−1) and negligible hemolytic activity, which ensure their good accumulation in tumor tissues, good stability against dilution in circulation and good blood compatibility, respectively. Moreover, the prepared FA-PEG/PEO-PPO-PCL micelles have obvious sustained release behavior in vitro. For cellular uptake study, the mixed micelles are discovered to be more effective to internalize DTX in FRs-positive MCF-7 cells via FRs mediated endocytosis compared with PEO-PPO-PCL micelles. Furthermore, the in vitro cytotoxicity indicated that active-targeting mixed micelles allowed a more significant inhibition of MCF-7 cells growth. Thus, the as-prepared DTX-loaded FA-PEG/PEO-PPO-PCL micelles provide a promising combined therapeutic strategy for antitumor therapy. Acknowledgements We gratefully acknowledge the financial support from National Natural Science Foundation of China (NSFC, No. 21373126), the Natural Science Foundation of Shandong Province, China (ZR2017MB045) and the Fundamental Research Funds for the Central Universities and the Scholarship of China Scholarship Council (16CX05013A). References [1] A. Singh, M. Talekar, T.H. Tran, A. Samanta, R. Sundaramb, M. Amiji, Combinatorial approach in the design of multifunctional polymeric nano-delivery systems for cancer therapy, J. Mater. Chem. B 2 (2014) 8069–8084. [2] A. Longhi, C. Errani, M. De Paolis, M. Mercuri, G. Bacci, Primary bone osteosarcoma in the pediatric age: state of the art, Cancer Treat. Rev. 32 (2006) 423–436. [3] T. Shimada, M. Ueda, H. Jinno, Development of targeted therapy with paclitaxel incorporated into EGF-conjugated nanoparticles, Anticancer Res. 29 (2009) 1009–1014. [4] D. Wang, C. Richter, A. Ruhling, S. Huwel, F. Glorius, H.J. Galla, Anti-tumor activity and cytotoxicity in vitro of novel 4,5-dialkylimidazolium surfactants, Biochem. Biophys. Res. Commun. 467 (2015) 1033–1038. [5] C.P. Leamon, Folate-targeted drug strategies for the treatment of cancer, Curr. Opin. Investig. Drugs 9 (2008) 1277–1286. [6] N. Kolishetti, S. Dhar, P.M. Valencia, L.Q. Lin, R. Karnik, S.J. Lippard, Engineering of self-assembled nanoparticle platform for precisely controlled combination drug therapy, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 17939–17994. [7] Y.D. Livney, Y.G. Assaraf, Rationally designed nanovehicles to overcome cancer chemoresistance, Adv. Drug Deliv. Rev. 65 (2013) 1716–1730. [8] I.G. Campbell, T.A. Jones, W.D. Foulkes, J. Trowsdale, Folate-binding protein is a marker for ovarian cancer, Cancer Res. 51 (1991) 5329–5338. [9] M.D. Salazar, M. Ratnam, The folate receptor: what does it promise in tissuetargeted therapeutics, Cancer Metastasis Rev. 26 (2007) 141–152.

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