Cyclic RGD targeting cisplatin micelles for near-infrared imaging-guided chemotherapy

Abstract

Nowadays imaging-guided chemotherapy is of great importance for developing highly efficient nanomedicines for cancer therapy. However, drug accumulation and release in vivo cannot be tracked in most studies due to the imaging moiety. In this study, near-infrared (NIR) dyes, cisplatin and dextran were used to prepare an imaging-guided chemotherapy drug with a cyclic peptide arginine-glycine-aspartic acid (RGD) targeting property for the treatment of breast cancer. Confocal laser scanning microscopy (CLSM) analysis indicated that the cisplatin-loaded micelles with cRGD decoration were quickly taken up by breast cancers cells in vitro according to the fluorescence of the NIR dye. More importantly, in vivo bioluminescence imaging analysis indicated that the drug-loaded micelles were preferentially distributed in the tumor regions of tumor-bearing mice. Furthermore, combining RGD targeting, in vivo imaging and therapeutic properties, this nanomedicine was suggested to have a good anti-tumor activity in vivo. Our study highlights the potential of combining active targeting and NIR fluorescence imaging in one drug delivery system for cancer diagnosis and therapy.

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1 Introduction

Multifunctional nanoparticles for tumor imaging, cell targeting and anti-cancer drug delivery have attracted a great deal of attention for cancer therapy.^1–4 Nanoparticles have been designed to be equipped with several moieties to optimize the treatment of cancer. For example, targeting moieties can improve drug accumulation in certain tumors, imaging moieties can be used to analyze the drug biodistribution in vitro or in vivo, and microenvironmental sensors (pH, redox, oxygen, proteases, and temperature) can trigger drug release in tumors reducing the damage of normal tissues.^5–7 These nanoparticles can not only selectively accumulate in certain tumors that can be observed via imaging moieties, but also release chemotherapy drugs to cancer cells to perform a therapeutic function.^8–11 These all-in-one systems hold great promise for cancer therapy.

Polymeric micelles have attracted widespread interest in anti-cancer drug delivery because of their hydrophobic core.^12–16 Until now, various types of materials have been used to prepare block copolymers which can self-assemble into nanoparticles for drug delivery.^17–19 Among these materials, dextran has been considered as a good candidate due to its excellent biocompatibility and degradability.^20,21 By introducing a hydrophobic building block, the drug-loaded dextran micelles showed enhanced cell uptake, and superior antitumor activity compared to small molecular drugs.^22–24

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY, BDP) has been widely used in bioimaging and diagnostics due to its excellent characteristics including environmentally robust fluorescence, high photostability and brightness, and favorable cell permeability.^25–28 BODIPY dyes whose emission maxima are located in the far-red and near-infrared (NIR) (650–900 nm) are preferred in biomedical imaging because of their deep tissue penetration and high signal-to-noise ratio.^29,30 In this study, NIR BODIPY (E_xmax = 590 nm, E_mmax = 673 nm) grafted dextran was used to prepare micelles for imaging-guided chemotherapy. The drug distribution and release could be obtained in real-time using an optical imaging system. In addition, cyclic peptide arginine-glycine-aspartic acid (RGD) which could selectively bind with α_vβ₃ and α_vβ₅ integrins was utilized to deliver cisplatin (CDDP) to breast cancers.^31–33 Cisplatin, a famous antitumor drug widely employed in cancer chemotherapy, was loaded through complexing with the carboxyl groups of dextran.^34–36

In this study, the CDDP release, the cellular uptake and cytotoxicity of nanoparticles in vitro, and the imaging and treatment of cancer in vivo were evaluated in detail.

2 Material and methods

2.1 Synthesis of near-infrared BODIPY dye

The synthesis of BODIPY has been reported by our laboratory.³⁷ Its chemical structure is shown in Scheme 1. The UV-Vis absorption and fluorescence emission were tested and found to be 643 and 673 nm in dimethylformamide (DMF).

Scheme 1 (A) The chemical structure of NIR BODIPY and RGD; (B) the synthesis of dextran-NIR and dextran-RGD; (C) preparation of cisplatin micelles.

2.2 Synthesis of dextran derivatives with succinic anhydride (dextran-SA)

Dextran (1 g, 6.17 mmol) was dissolved in 6 mL of dry DMSO in a flame-dried flask, followed by addition of 4-(dimethylamino) pyridine (DMAP, 754 mg, 6.17 mmol) solution in DMSO (2 mL) and succinic anhydride (SA, 617 mg, 6.17 mmol) in DMSO (2 mL), respectively. The reaction was performed at 50 °C for 24 h under nitrogen. The product was isolated by precipitation in cold ethanol, washed several times with ethanol, and dried under vacuum. The resulting white powder was then dissolved in deionized water, and dialyzed for 48 h to remove the excess reactants. The final product was obtained as a white powder after lyophilization (yield: 96%).

2.3 Synthesis of dextran-RGD and dextran-BDP

For dextran-RGD synthesis, 500 mg (50 μmol) dextran-SA, 14.4 mg (75 μmol) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 16.3 mg (75 μmol) N-hydroxysulfosuccinimide (Sulfo-NHS) were dissolved in 10 mL deionized water, and the pH value of the solution was adjusted to 5.4. The reaction was conducted at room temperature for 2 h and then 30 mg (50 μmol) RGD peptide was added. After 12 h of reaction, the solution was dialyzed against distilled water for 12 h (MWCO = 3500), the final suspension in a dialysis bag containing RGD-dextran was freeze-dried. The RGD peptide content of the polymers was determined by measuring the arginine content as reported in ref. 38.

Dextran-BDP was synthesized as below. 100 mg of dextran (0.38 mmol COOH) was dissolved in 10 mL of DMSO, and 3.6 mg of EDC·HCl (2.4 mg) and 1.4 mg of NHS were added at 0 °C and stirred for 12 h, then 9.5 mg of BDP (0.012 mmol) was added and stirred for an additional 12 h. The reaction product was dialyzed against distilled water for 12 h (MWCO = 3500) and freeze-dried. The BDP content of the obtained dextran-BDP was determined using a UV-Vis spectrometer.

2.4 Preparation of BDP conjugated dextran–cisplatin complex micelles (M(BDP/Pt)) and RGD decorated micelles (RGD-M(BDP/Pt))

The synthesis of cisplatin–dextran complex micelles has been described in the literature.³⁹ 0.1 g of cisplatin was suspended in 10 mL distilled water and mixed with 0.113 g silver nitrate (AgNO₃/cisplatin = 2 mol mol⁻¹). After stirring in the dark for 4 h at room temperature, the AgCl precipitates were removed by filtration. The filtrate was used directly. The mixture of dextran-SA and dextran-BDP (40 mg with weight ratio of 1 : 1) was dissolved in 4 mL deionized water, and then 7.5 mg Na₂CO₃ was added to deprotonate the carboxyl groups. Different amounts of silver nitrate were added to the solution and stirred for 2 h in the dark, and then the solution was dialyzed (cutoff = 3500) against deionized water for 10 h to remove unreacted cisplatin. The obtained micelles are abbreviated as M(BDP/Pt).

RGD decorated micelles were prepared similarly by adding 20 wt% dextran-RGD to the mixture polymer for the drug complex. These micelles are abbreviated as RGD-M(BDP/Pt).

The BDP content of the micelles was measured using a UV-Vis spectrometer and the cisplatin-loading content (LC) and encapsulation efficiency (EE) were determined by ICP-OES. The LC and EE were calculated according to the following formula:

LC (%) = weight of loaded drug × 100/weight of loaded drug + weight of polymer used;

EE (%) = weight of loaded drug × 100/weight of feeding drug.

2.5 Characterization of polymer micelles

The morphology of the polymer micelles was determined using a transmission electron microscope (TEM) (JEOL JEM-1011 electron microscope). The size and size distribution of the micelles were measured by dynamic light scattering (DLS) with a vertically polarized He–Ne laser (DAWN EOS, Wyatt Technologies). The scattering angle was fixed at 90° for DLS measurement at 25 °C.

2.6 Cell viability assay

Mouse mammary carcinoma (EMT6) cells were seeded in 96-well plates at a density of 4 × 10³ cells per well in 100 μL Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) for 12 h at 37 °C in 5% CO₂. The medium was replaced with 200 μL DMEM medium containing different concentrations of polymers (dextran-SA : dextran-BDP = 1 : 1) ranging from 100 to 1000 μg mL⁻¹ for 48 h. 20 μL MTT (5 mg mL⁻¹) was added to each well for 4 h incubation, and then 150 μL DMSO was added to each well to dissolve the blue formazan formed in the live cells. The absorbance was measured on a microplate reader (BioTek, EXL808) at 490 nm. Experiments were repeated three times.

2.7 In vitro cisplatin release

The release profiles of cisplatin from M(BDP/Pt) micelles at different pH values (pH 7.4 and pH 5.0) were tested using a dialysis method. Briefly, the weighted freeze-dried M(BDP/Pt) was suspended in 5 mL of release medium and transferred into a dialysis bag (cutoff = 3500) and sealed. The release experiment was initiated by placing the sealed dialysis bag into 20 mL of release medium (20 mM PBS, pH 7.4 and pH 5.0) at 37 °C with shaking. At selected time intervals, 1 mL of release media was taken out and replenished with an equal volume of fresh media. The amount of Pt released was measured by ICP-OES, and the cumulative Pt release percentage was calculated and plotted against the release time.

2.8 Subcellular localization in cancer cells

The subcellular localization of the M(BDP/Pt) micelles and RGD-M(BDP/Pt) micelles was observed by confocal laser scanning microscopy (CLSM). Briefly, 1 × 10⁵ EMT6 cells per well were seeded onto cover slides in a six-well plate and incubated for 12 h. The medium was replaced with 2 mL fresh medium containing 20 μg BDP for 1, 3, 6, 9, 12, and 24 h, respectively. The cells were washed three times with PBS, and fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature. The cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for the cell nucleus. The cellular localization was visualized under a confocal laser scanning microscope (Carl Zeiss LSM 780).

In order to investigate whether dextran-NIR micelles escaped from endosomes, a late endosome and lysosome specific probe was used for co-localization analysis. The cell seed and fixed process was the same as mentioned above. After fixing with 4% (w/v) paraformaldehyde, the cells were stained with LysoTracker Red (Beyotime Institute of Biotechnology, 50 nM) for 30 min and washed with PBS three times. DAPI was excited with a blue diode (405 nm) and detected in the 415–460 nm range. LysoTracker was excited with a laser (555 nm) and detected in the 570–590 nm range. NIR BODIPY was excited at 633 nm and detected in the 650–775 nm range. CLSM images were captured via a confocal microscope under the same parameters.

2.9 Cytotoxicity of M(BDP/Pt) and RGD-M(BDP/Pt)

A cytotoxicity test was conducted to examine the antitumor activity of free cisplatin, M(BDP/Pt) and RGD-M(BDP/Pt) via 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, 4000 cells per well were seeded to a 96 well plate and incubated for more than 12 h. The medium was refreshed with 200 μL DMEM medium containing different concentrations of Pt or micelles for 48 h. The Pt concentration ranged from 0.01 to 50 μg mL⁻¹. 20 μL MTT (5 mg mL⁻¹) was added to each well and incubation was continued for more 4 h, and then 150 μL DMSO was added to each well to dissolve the blue formazan formed in the live cells. The absorbance was measured on a microplate reader at 490 nm. Experiments were repeated three times.

2.10 Biodistribution of micelles in vivo

All animal experiments were performed in compliance with the guidelines established by the Animal Care and Use Committee of Beihua University, and all procedures were approved by the Animal Care and Use Committee of Beihua University. Ten BALB/C mice were utilized to implant a xenograft EMT6 mouse mammary carcinoma. To develop the tumor xenograft, 1 million EMT6 cells were injected into the lateral aspect of the anterior limb of the mice. After the tumor volume reached 50–100 mm³, xenograft-bearing mice were divided randomly into 2 groups: M-(BDP/Pt) and RGD-M(BDP/Pt) micelle groups. M-(BDP/Pt) or RGD-M-(BDP/Pt) micelles were injected into xenograft-bearing mice via a tail vein (with an equivalent BDP dose of 30 μg). The fluorescence observations were carried out using an imaging system 8, 12 and 24 h after tail vein injection of the micelles. The imaging system (CRI Maestro 500FL) used in this study consisted of a light-tight box equipped with a 150 W halogen lamp and an excitation filter system (575–605 nm) to excite NIR BODIPY. Fluorescence was detected using a CCD camera equipped with a C-mount lens and an emission filter (longpass: 645 nm cut-in).

2.11 Anti-tumor efficacy

All animal experiments were performed in compliance with the guidelines established by the Animal Care and Use Committee of Beihua University, and all procedures were approved by the Animal Care and Use Committee of Beihua University. The in vivo anti-tumor efficacy of the drug-loaded micelles was evaluated using the xenograft EMT6 mouse mammary carcinoma implanted on the BALB/C mice. When the tumor volumes of the mice reached 30–70 mm³, this day was designated as day 0. The mice were weighed and randomly divided into 4 groups (5 mice per group): saline, free cisplatin, M-(BDP/Pt) and RGD-M-(BDP/Pt) micelles (dosage: 5 mg Pt equivalent per kg body weight). The drug injection was carried out on days 0, 2, and 4 via the tail vein. The anti-tumor efficacy evaluation was assessed by measuring the tumor volume. The anti-tumor activities were evaluated by measuring the tumor volume (V) estimated by the following equation: V = ab²/2, where a and b stand for the major and minor axes of the tumor measured via a vernier micrometer.

2.12 Statistics

All experiments were performed at least three times and all results are expressed as mean ± SD (standard deviation). Student’s t-test was used to demonstrate statistical significance (P < 0.05).

3 Results and discussion

To simultaneously endow micelles with active targeting and bioimaging properties, cyclic RGD peptide and BODIPY dye were conjugated to the dextran in this study. The chemical structures of BODIPY and RGD are shown in Scheme 1(A). Scheme 1(B) shows the synthesis process for dextran-RGD and dextran-NIR and Scheme 1(C) is a schematic illustration of dextran-RGD targeting cisplatin micelle complexes.

3.1 Modification of dextran

The synthesis of dextran-SA was performed according to the literature with high yield (>95%). The ¹H NMR spectra of dextran and dextran-SA are shown in Fig. 1. The introduced side carboxyl groups provide sites for further functionalization.

Fig. 1 ¹H NMR spectra of dextran and dextran-SA.

We had previously synthesized a highly hydrophobic BODIPY dye and investigated its off–on fluorescence properties by conjugating it with mPEG to form nanovesicles.³⁷ Similarly, in this paper, we conjugated BODIPY onto dextran side groups and formed dextran micelles with off–on fluorescent properties. The BODIPY moieties in the micelles are in an aggregated state and thus fluoresce weakly due to the aggregation quenching effect. Once the micelles enter into the cells and the micelles dissociate, the NIR dyes become intensely fluorescent.³⁷ The synthesis process of dextran-NIR is shown in Scheme 1(B) and the BODIPY content in dextran-NIR was determined to be 6.7% (w/w) using a UV-Vis spectrometer.

The cyclic RGD was selected as an active targeting moiety and was conjugated to dextran as shown in Scheme 1(B). The molar ratio of cRGD peptide to dextran was determined to be 56.4% by measuring the arginine content using the reported method.³⁸

3.2 Preparation of M(BDP/Pt) and RGD-M(BDP/Pt) micelles

The residual carboxyl groups in dextran-RGD and dextran-NIR can be used to coordinate with cisplatin. Three kinds of polymers (dextran-SA, dextran-RGD, and dextran-NIR) were used to prepare micelles. With the increase of the CDDP feed ratio, the DLC and mean diameter of the micelles increased gradually, while the zeta potentials decreased, which may be attributed to the consumption of carboxyl groups while complexing with cisplatin. To find a balance between the CDDP loading and the size of polymer micelles, in this study 15% CDDP was used to prepare our all-in-one micelles.

Functionalized micelles were prepared by adding dextran-RGD or dextran-NIR or both of them to the polymer. 20 wt% of RGD conjugated polymer in the mixture was selected according to our previously study. The prepared micelles were characterized by TEM and DLS as shown in Fig. 2. The results showed that all micelles displayed a spherical structure. The diameters of M(Pt), RGD-M(Pt), M(BDP/Pt) and RGD-M(BDP/Pt) were 48, 55, 169 and 177 nm, respectively. After adding RGD decoration, the particle sizes increased about 6 or 7 nm.

Fig. 2 Characterization of different polymer micelles by TEM (A) and DLS (B) analysis. (a): M(Pt); (b): RGD-M(Pt); (c): M(BDP/Pt); (d): RGD-M(BDP/Pt). Scale bar: 500 nm.

3.3 Drug release in vitro and biocompatibility analysis

The released Pt from M(BDP/Pt) was investigated in PBS buffers (pH 5.0 and pH 7.4). The released Pt was determined using ICP-OES. As shown in Fig. 3A, a sustained release of Pt was observed in PBS buffers within 120 h. However, 63% of the Pt was released in pH 5.0 PBS at 120 h, while only a 25% Pt release was detected in pH 7.4 PBS. The biocompatibility of nanomaterials is very important for their biomedical application.⁴⁰ In vitro biocompatibility tests of Dex-SA/NIR (Dex-SA : Dex-NIR = 1 : 1) against EMT6 cells for 48 h were performed at different concentrations. As shown in Fig. 3B, the cell viability was more than 82% at all test concentrations up to 1 mg mL⁻¹, indicating good compatibility of the polymers with the EMT6 cells.

Fig. 3 Drug release in vitro, cell viability analysis and cytotoxicity of Pt-loaded micelles. (A): In vitro cumulative release profiles of Pt from M(BDP/Pt) at different pHs (pH 5.0 and 7.4) at 37 °C; (B): biocompatibility assay of polymers (Dex-SA/NIR); (C) and (D): the cytotoxicity of CDDP, M(BDP/Pt) and RGD-M(BDP/Pt) were measured after incubation with EMT6 cells for 48 (C) and 72 h (D) by MTT assays. All the results are an average of three measurements, and given as mean ± SD.

In order to examine the cytotoxicity of our micelles, MTT analyses were performed on the EMT6 cells at different concentrations (0.01–10 μg mL⁻¹). The survival rates of EMT6 cells after incubation with free CDDP, M(BDP/Pt) and RGD-M(BDP/Pt) for 48 h were examined. As shown in Fig. 3C and D, the cell viability of cells treated with RGD-M(BDP/Pt) was much lower than that of cells treated with M(BDP/Pt) at most drug concentrations, which may be ascribed to the much greater cell uptake of RGD-decorated micelles than the untargeted micelles. Moreover, 67% of the cells treated with RGD-M(BDP/Pt) were killed at a concentration of 10 μg mL⁻¹, which was even higher than the 60% for free CDDP. These results suggest that our dextran micelles have good biocompatibility and stronger cytotoxicity than free CDDP.

3.4 Cellular uptake

The cellular uptake of RGD-M(BDP/Pt) and M(BDP/Pt) was examined by measuring the fluorescence of BDP (excitation wavelength: 633 nm) by CLSM. As shown in Fig. 4, the fluorescence could not be observed until after 6 h of incubation for M(BDP/Pt) and the fluorescence intensity from BODIPY increased gradually by prolonging the incubation time from 6 h to 24 h. As for RGD-M(BDP/Pt) micelles, BDP fluorescence could be detected after 3 h of incubation (not shown), and appeared even brighter than that in EMT6 cells incubated with M(BDP/Pt) for 6 h. As reported by many reports,⁴¹ RGD targeted nanoparticles could be internalized via receptor mediated endocytosis, which is much faster than for untargeted controls. So the faster BDP release in the RGD-M(BDP/Pt) group may be attributed to the different pathways of endocytosis between M(BDP/Pt) and RGD-M(BDP/Pt).

Fig. 4 CLSM images of the EMT6 cells after 1, 6, 12 and 24 h of incubation with M(BDP/Pt) (A) or RGD-M(BDP/Pt) (B). The cellular nuclei were stained by DAPI (blue), BODIPY fluorescence is indicated as red, BF represents the bright field, and Merge is the overlay of the three images.

The above results indicated a positive correlation between fluorescence intensity and incubation time for both micelles. Considering the self-quenching properties of our NIR BDP at high concentration, the higher fluorescence intensity could be evidence of more BDP release from micelles.

3.5 Co-localization with lysosomes

To investigate whether micelles escaped from endosomes, the late endosomes and lysosomes were stained by LysoTracker in this study. As shown in Fig. 5, merging of the yellow fluorescence could be observed after 6 h incubation with M(BDP/Pt) or 3 h with RGD-M(BDP/Pt). The merged yellow fluorescence clearly demonstrated co-localization of micelles with endosomes, suggesting potential endosome escape. As the incubation time increased to 24 h, the intensity of the yellow fluorescence reached a maximum. These results indicated that RGD-M(BDP/Pt) efficiently enhanced the endosomal escape in EMT6 cells compared with M(BDP/Pt).

Fig. 5 CLSM images of EMT6 cells incubated for 6, 9, 12 and 24 h with M(BDP/Pt) (A) or RGD-M(BDP/Pt) (B). The late endosomes and lysosomes were stained with LysoTracker (green). The cell nuclei were stained by DAPI (blue), and the red fluorescence was emitted by BDP. The yellow fluorescence, which was generated by overlaying green and red fluorescence is indicated as arrows.

3.6 Distribution of micelles in vivo

To examine the distribution of M(BDP/Pt) and RGD-M(BDP/Pt) in vivo, equal BDP doses of micelles (30 μg) were administered via tail vein injection. As shown in Fig. 6, for the M(BDP/Pt) group fluorescence in the tumor could not be detected until 48 h after injection, and the peak intensity of fluorescence was observed 168 h after injection; however, a high BDP fluorescence in mice injected with RGD-M(BDP/Pt) could be detected within 24 h of injection, and the fluorescence reached its maximum at 96 h after injection. These results indicated that the BODIPY release from M(BDP/Pt) micelles was delayed for 24 h to 48 h compared with that of RGD-M(BDP/Pt) micelles. The delayed BODIPY release of M(BDP/Pt) in vivo is consistent with the results of BODIPY release in vitro. The significant difference in NIR BODIPY release between M(BDP/Pt) and RGD-M(BDP/Pt) could be associated with the different pathway of endocytosis. Many reports have shown that receptor mediated endocytosis accelerated tumor cell uptake of RGD-decorated micelles.^31–33

Fig. 6 BDP release in vivo was visualized after tail vein injection using a CRI Maestro 500FL imaging system.

3.7 Anti-tumor efficacy

The anti-tumor activity was tested on xenograft EMT6 mouse mammary carcinoma. The tumor volume was measured and recorded for 12 days. As shown in Fig. 7, compared with saline, free CDDP and CDDP-loaded micelles showed obvious anti-tumor efficacy, and both micelle groups showed better tumor inhibition. The cRGD decorated Pt-loaded micelles resulted in the best efficacy of tumor inhibition. At day 12, free CDDP, M(BDP/Pt) and RGD-M(BDP/Pt) had tumor suppression rates of 39.1%, 68.2% and 76.4%, respectively. The better anti-tumor efficacy of RGD-M(BDP/Pt) compared to untargeted micelles could be attributed to the greater cellular uptake of Pt-loaded micelles as indicated by the cell experiment in vitro.

Fig. 7 In vivo anti-tumor efficacy evaluation of the drug-loaded micelles on the EMT6 xenograft. The Pt injection was carried out on days 0, 2, and 4 via the tail vein at a dose of 5 mg Pt equivalent per kg body weight. Differences between groups were evaluated by analysis of variance (ANOVA) to demonstrate statistical significance (P < 0.05).

4 Conclusion

In this study, multifunctional micelles were successfully developed by combining RGD targeting, near-infrared BODIPY imaging, and anticancer drug delivery. The BODIPY and RGD were conjugated to the pendant groups of dextran and the anticancer drug cisplatin was complexed with the pendant carboxyl groups of dextran. CLSM and live animal imaging analysis indicated preferred accumulations of dextran micelles in EMT6 breast cancer cells and breast cancer xenografts after cRGD decoration. Moreover, the dextran micelles displayed good anti-tumor efficacy in vivo. This work highlights the potential role of multifunctional theranostic micelles in tumor diagnosis and cancer therapy.

Acknowledgements

Financial support was provided by the Science and Technology Department of Jilin Province (Project no. 20111809, 20130624003JC, 2014866 and 20130206050YY), and National Natural Science Foundation of China (Project no. 81170632, 91227118, 51503003 and 51373167).

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