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Fluorescent Organic Nanoparticles Constructed by a Facile “Self-Isolation Enhanced Emission” Strategy for Cell Imaging Dongfeng Dang, Xiaochi Wang, Daquan Wang, Zhiwei Yang, Dongxiao Hao, Yanzi Xu, Shengli Zhang, and Lingjie Meng ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00409 • Publication Date (Web): 27 Apr 2018 Downloaded from http://pubs.acs.org on April 27, 2018

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ACS Applied Nano Materials

Fluorescent Organic Nanoparticles Constructed by a Facile “Self-Isolation Enhanced Emission” Strategy for Cell Imaging Dongfeng Dang,a,‡ Xiaochi Wang,a,‡ Daquan Wang,a Zhiwei Yang,a Dongxiao Hao,a Yanzi Xu,a Shengli Zhang,a Lingjie Menga,b*

a

School of Science, MOE Key Laboratory for Nonequilibrium Synthesis and Modulation of Condensed Matter, Xi'an Jiaotong University, Xi'an 710049, P. R. China. b

Instrumental Analysis Center, Xi'an Jiaotong University, Xi'an 710049, P. R. China.

Corresponding Authors: Prof. Lingjie Meng E-mail: [email protected] (L.J. Meng) ‡

Dongfeng Dang and Xiaochi Wang contributed equally to this manuscript.

KEYWORDS: Fluorescent organic nanoparticles; Quenching; Photo-bleaching; Self-Isolation Enhanced Emission (SIEE); Cellular imaging.

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ABSTRACT: To achieve the highly emissive features and overcome the troublesome photobleaching for fluorescent organic molecules, a facile and versatile strategy named “SelfIsolation Enhanced Emission (SIEE)” was developed to prevent the π-π stacking of organic fluorophores by linking alkyl chains on their conjugated backbones. As a proof-of-concept, one or two octyl groups were grafted onto the backbone of 4,7-di(thiophen-2-yl)benzo [c][1,2,5]thiadiazole (termed as DTBT-0), resulting in two different molecules, termed as DTBT1 and DTBT-2, respectively. Compared with DTBT-0, DTBT-1 and DTBT-2 exhibited remarkably enhanced fluorescent properties in both aggregated thin films and nanoparticles, demonstrating that the SIEE method could isolate the fluorophores effectively and then prevent their π-π stacking to achieve the impressive fluorescent properties. After proper surface modification, excellent water dispersibility, biocompatibility and improved resistance to photobleaching were also achieved for highly emissive DTBT-2 based nanoparticles, which were then successfully applied for cellular imaging.


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1. Introduction Over the past few decades, semiconductor quantum dots (QDs),1-2 rare earth-based nanoparticles,3-4 carbon dots (CDs),5-6 and organic molecule-based fluorescent nanoparticles (FONPs) 7-14 have attracted great attentions for their wide range of applications in biosensors and biomedical imaging.15-16 However, while QDs and rare earth nanoparticles potentially exhibit cytotoxicity due to their heavy metal ions,17 red and near-infrared fluorescent CDs for wholebody fluorescence imaging are difficult to synthesize. Meanwhile, although FONPs usually exhibit good chemical stability and tailorability, easily-tunable particles size, high quantum yield, and excellent biocompatibility,18-21 the main challenge in currently developed fluorescent molecules and/or their corresponding FONPs is the inevitable fluorescence quenching in aggregated states due to the notorious aggregation-caused quenching (ACQ) effect.22 To overcome this problematic ACQ phenomenon in organic fluorescent molecules, Tang et al. discovered an uncommon phenomenon, namely “aggregation-induced emission” (AIE), in which weak or no emission was observed from molecules in dilute solutions, but high luminescence was achieved when they were aggregated in highly concentrated solutions or thin films.23-25 Based on this unique nature for AIE, the developed AIE luminogens (AIEgens) always exhibited the impressive emission properties in the solid states, which is the fundamental property to monitor the dynamic changes in cells by fluorescent imaging.26-27 Additionally, the emission color can also be fine-tuned for these developed AIEgens through the modification of molecular structures, indicating their great potentials not only for the imaging in vitro, but also in vivo.28-29 It’s also interesting that some water soluble AIEgens can make this fluorescent imaging much more easier through a wash-free approach.30 All these demonstrated that the fantastic discovery of AIE has opened a new chapter in exploration of fluorescent molecules and FONPs in


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biomedical imaging.31-35 Furthermore, another strategy, which “isolate” and “fasten” fluorescent moieties into a stable cross-linked polyphosphazene (PPZ), was proposed by our research group. The strategy has also been demonstrated to work well in overcoming the ACQ effect.36-38 Although these two methods can effectively suppress the ACQ effect, careful molecular design and synthesis are always needed to obtain special molecular structures. For example, non-planar structures containing several molecular rotors are necessary for the AIE method, whereas complicated cross-linking reactions are required in the “isolation” and “fasten” strategy. On the other hand, it’s well demonstrated that alkyl chains are not only generally introduced to improve the solubility of conjugated fluorescent molecules, but also can affect their intermolecular interactions, packing modes, and even molecular conformations significantly,39-42 and thus their optical and optoelectronic properties in solid states could be fine-tuned.43-45 However, it is worth noting that these well investigated fluorescent molecules in this case are usually the twisted molecules with weak emission-quenched π-π stacking in solid states,46-49 few studies were focused on the ACQ molecules to further enhance their emission efficiency through alkyl chains.50 Also, the aggregated states for organic fluorescent molecules included both the aggregated solid states and aggregated nanoparticles. However, all these highly emissive fluorescent molecules through alkyl chains in molecular backbones were only well studied in solid states through various measurements,51-52 their corresponding fluorescent nanoparticles widely used in biomedical imaging were seldom investigated. Based on these considerations, we proposed a rather facile and versatile strategy here to isolate the ACQ fluorophores through the alkyl chains in molecular backbone, namely “SelfIsolation Enhanced Emission (SIEE)”. Based on this approach, we speculated that the electron and energy transfers between fluorophores could effectively be suppressed when alkyl chains


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were grafted, and thereby prevent the π-π stacking, leading to the highly emissive fluorescent molecules and their corresponding FONPs with proper size, good chemical stability, and also excellent biocompatibility for biomedical imaging. As a proof-of-concept, octyl groups were grafted onto the fluorescent backbone of 4,7-di(thiophen-2-yl)benzo[c][1,2,5]thiadiazole (DTBT0) to afford DTBT-1 and DTBT-2, respectively (Scheme 1). It’s interesting that, in contrast to DTBT-0, DTBT-1 and DTBT-2 in both aggregated thin film and nanoparticles exhibited remarkably enhanced fluorescent properties. Also molecular dynamics (MD) simulations indicated that, through the SIEE strategy, the interactions between chromophores were prevented by alkyl chains in the aggregated nanoparticles, and therefore, effectively reduced the π-π stacking while significantly improved the fluorescent properties. Finally, after modification with biocompatible molecules, good water dispersibility, biocompatibility, and also improved photobleaching resistance were achieved for DTBT-2-based FONPs. Accordingly, they were successfully applied in cellular imaging.

2. Experimental Section 2.1 Chemicals All chemicals were analytical grade and used without further purification, unless otherwise stated. Polyethylene glycol (PEG, 2k), carboxylated chitosan (CCHI, 200 mpa.s), and polymine (PEI, 10 k) were purchased from Aladdin Chemical Reagent Co., Ltd. Fetal bovine serum (FBS) and high glucose Dulbecco’s Modified Eagle’s medium (DMEM) were purchased from Hyclone. WST-1 cell viability assay kit was purchased from Beyotime Biotechnology Co., Ltd. Ultra-pure water (18.2 MΩ·cm−1) obtained from a Millipore Milli-Q purification system was used throughout the experiments.


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2.2 Characterization and measurement All 1H NMR and

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C NMR spectra were recorded on a Bruker Advance 400 MHz using

CDCl3 as the solvent. Mass Spectrometer were measured using the WATERS I-Class VION IMS QTof. The Ultraviolet-Visible (UV-Vis) spectra were recorded using a SHIMADZU UV-2550 spectrophotometer. The photo-luminescence (PL) spectra were collected on an Edinburgh Instrument FLS980 fluorescence spectrophotometer. The aggregated thin films based on DTBT0, DTBT-1, and DTBT-2 for PL measurements here were prepared by spin-coating using their corresponding tetrahydrofuran (THF) solution with a concentration of 10 mg/mL. The absolute quantum yields were obtained on an Edinburgh Instrument FLS980 Integrating sphere. The morphology and size of FONPs were monitored via HR-TEM (JEOL-2100). The TEM samples were prepared by air-drying the FONPs water dispersion overnight. The hydrodynamic sizes and distributions, and the zeta-potentials (ζ) were measured by a Malvern Zetasizer Nano ZS90 at room temperature. Cell cytotoxicity was determined using a MD Spectra Max 190 microplate reader. Confocal laser scanning microscopy was carried out on a Zeiss LSM700 confocal laser scanning microscope. 2.3 Preparation and Functionalization of FONPs FONPs were prepared using common nano-precipitation method. Briefly, fluorescent molecules solutions (DTBT-0, DTBT-1, and DTBT-2) in THF of various concentrations were quickly added into ultra-pure water (5 mL) under a vigorous stirring at room temperature. The mixture was let stand for 1 h at room temperature to allow evaporation of THF and to stabilize the nanoparticles. Functionalizations of FONPs were carried out by similar method; however,


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pure water was replaced by 0.5 mg·mL-1 aqueous polymers, which include PEG, CCHI, and PEI.53 2.4 Preparation of the dataset and Molecular dynamics (MD) simulations Initial models were generated by packmol according to the previously published procedure.54 Topology file of each molecule was then processed with the leap and antechamber modules in AMBER11 utilizing the recommended parameters

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and the AM1-BCC charges.56

After that, the TIP3P models were employed for water molecules.57 Conventional MD simulations were performed using a well-established protocol, in agreement with our past works.58 Briefly, following bad contact removals by energy minimizations and system equilibration by position-restrained MD simulations, each system underwent a 100 ns MD simulation in an isothermal and isobaric ensemble (NPT, T= 300 K and P= 1 atm) with periodic boundary conditions. The particle-mesh Ewald method was imposed in long-range electrostatics with a 10 Å non-bonded cut-off, and the SHAKE algorithm was performed to constrain bonds of hydrogen. MD trajectories were saved every 10.0 ps, each with an integration time step of 2.0 fs. 2.5 Cytotoxicity assay and Confocal laser scanning microscopy of FONPs Cell cytotoxicity was determined using a standard WST-1 assay. HeLa and H9C2 cells were seeded in 96-well plates at a density of 5×104 per well for 24 h at 37 °C under 5% CO2 atmosphere. Cells were then treated with various concentrations of FONPs and functionalized FONPs for 1 h. After that, the FONPs were discarded and replaced by fresh medium. After 24h incubation, 10 µL of WST-1 was added and cell viability was determined at 450 nm using a micro-plate spectrophotometer. HeLa and H9C2 cells were seeded in 24-well plates at a density of 5×104 per well for 24 h (37 °C, 5% CO2). FONPs were then added to the wells and incubated


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for 1 h. After that, the FONPs were replaced by fresh medium and further cultured for additional 24 h. Cellular imaging was performed using a laser scanning confocal microscope.

3. Results and discussion 3.1 Synthesis and Optical Properties Scheme 1. The molecular structures of DTBT-0, DTBT-1, and DTBT-2. Color code: gray (carbon); white (hydrogen); blue (nitrogen); and yellow (sulfur).

The molecular structures of DTBT-0, DTBT-1, and DTBT-2 were displayed in Scheme 1. The molecules were synthesized via the common Stille coupling reaction, from which a moderate yield of 70% was obtained (Scheme S1, Supporting Information). The synthesized molecules were characterized by 1H NMR,

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C-NMR and TOFF spectra. Their UV-Vis

absorption and PL spectra in THF solution were also determined. All molecules showed similar absorption spectra with two distinct absorption peaks at ~310 and ~460 nm corresponding to the π-π* transition and the intra-molecular charge transfer (ICT) interactions, respectively (Figure S1a, Supporting Information).59-61 These developed molecules also exhibited a distinct


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fluorescent peak at 600 nm (Figure S1b, Supporting Information). Interestingly, when octyl group was step-by-step introduced into the fluorescent backbone, red-shifts of 12-15 nm in both the absorption and emission spectra were observed. Moreover, these molecules exhibited a large stokes shift of 130 nm, associating with the minimization of cross-talk between excitation source and fluorescent emission. Such large stokes shifts can generally lead to the impressively high signal-to-noise ratios in various applications.62 PL spectra of these molecules in aggregated thin films (Thickness≈ 90 nm) were also investigated (Figure 1a). As illustrated, the fluorescent intensity was significantly enhanced when alkyl chains were presented in the conjugated molecular backbone. Then to further investigate their fluorescent properties of DTBT-0, DTBT-1, and DTBT-2, quantum yields (QY) in solution and thin films were also measured (Table 1). As anticipated, the absolute QY (over 40%) in solution measured by integrating sphere for all molecules were comparable, whereas that for DTBT-1 and DTBT-2 in aggregate states was remarkably increased. Compared with that for DTBT-0 (7%), the QY for DTBT-2 was as high as 32%, indicating that the octyl groups could effectively prevent the π-π stacking in conjugated chromophores, leading to high emission efficiency in the aggregate state. Moreover, compared to DTBT-0, DTBT-1 and DTBT-2 also exhibited a remarkably enhanced emission in the solid state (inset, Figure 1a), which is also consistent with the QY results. To describe the fluorescent properties of DTBT-0, DTBT-1, and DTBT-2, the transient decay spectra of their thin films were further examined (Figure 1b). The life-time (τ), radiative decay rates (kr), and non-radiative decay rates (knr) were also calculated and summarized (Table. 1). Compared with DTBT-0, DTBT-2 exhibited larger τ and kr, and also lower knr, thus had higher QY value.63 Interestingly, for DTBT-1, although lowest τ and largest knr values were observed caused by the larger dipole moment change for its asymmetric chemical


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structure, similar to that of DTBT-2, effectively reduced π-π stacking is observed to achieve the large kr value, finally leading to a moderate QY value here.

Figure 1. PL spectra (a) and Transient decay spectra (b) of DTBT-0, DTBT-1, and DTBT-2 in thin films. Table 1. Optical properties of fluorescent DTBT-0, DTBT-1, and DTBT-2. Compounds

[a]

Stokes shift QY [a] QY [b]

τ [b]

kr [c]

knr [d]

(×107 s-1) (×107 s-1)

(nm)

(%)

(%)

(ns)

DTBT-0

130

42.75

7.03

9.2

0.76

10.11

DTBT-1

134

44.99

16.02

5.4

2.97

15.54

DTBT-2

136

45.80

32.02

12.6

2.54

5.39

Measured in THF solution;

[b]

Measured in thin films;

[c]

Radiative decay rate in solid

state, kr =QY/τ; [d] Non-radiative decay rate in solid state, knr = 1/τ-kr. 3.2 Preparation and characterization of FONPs These fluorescent molecules exhibited good solubility in various common solvents, including THF, chloroform, dichloromethane, etc., but were insoluble in water. Therefore, the corresponding FONPs were obtained using nano-precipitation method, under which their THF solutions were added dropwise into water.64 To obtain FONPs with proper size for cell uptake,


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the concentration of fluorescent molecules in THF solution and also the solvent volume ratios (THF/Water) were fine-tuned here. With increasing DTBT-2 concentration, the hydrodynamic diameters gradually increased, while the polydispersity index (PDI) values decreased, and reached a value of 0.038 at DTBT-2 concentration of 0.2 mg·mL-1. The data also showed that they were mono-dispersed (Table S1 and Figure S2, Supporting Information). Similarly, with the increasing volume of DTBT-2 solution, the PDI values of FONPs decreased, and the hydrodynamic diameters gradually increased (Table S1 and Figure S3, Supporting Information). Based on these observations, 200 µL of DTBT-2 solution with a concentration of 0.2 mg·mL-1 was selected to prepare these FONPs. The particle size and also Zeta potentials were also listed and FONPs with proper size were finally achieved (Figure S4 and Table S2, Supporting Information). TEM images also showed that the nanoparticles prepared from all molecules were well dispersed with approximate diameters of 80-120 nm (Figure 2), indicating their great potential to be used in bio-imaging. Then the PL spectra for these developed FONPs were also recorded and distinct emissions between 550-650 nm were observed (Figure S5, Supporting Information). It’s also encouraging that under this optimized condition to prepare the nanoparticles, DTBT-2-based FONPs exhibited the highest fluorescence intensity, while DTBT0-based FONPs had the lowest fluorescence intensity. Then to further understand their different fluorescent properties, the emission intensity and also QY values for FONPs under the same concentration (molar concentration, [c]= 7.18×10-8 M) were investigated here (Figure S6, Supporting Information). As expected, similar to the results in solid states, the well-isolated nanoparticles based on DTBT-2 also exhibited the most impressive emission intensity. After that, the quantum yields for nanoparticles were calculated according to the following equation: QYs = QY 0 ×

FS A0 × ns 2 × F 0 AS × n0 2


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in which F is the integrated fluorescence intensity, A is the absorbance intensity at excitation wavelength, n is the refractive index for employed solvent, the subscript 0 and s represents reference compounds and prepared samples, respectively. Rhodamine 6G (R6G) in water was used as the reference (QY= 95%).65 As observed, DTBT-0 based nanoparticles just displayed a moderate QY values of 43.9%, however, when the SIEE methods were employed through alkyl chains in molecular backbone to afford FONPs based on DTBT-1 and DTBT-2, much higher QY values of 44.5% and 52.5% were finally achieved. These observations further indicated that as a result of the “Self-Isolation Enhanced Emission” strategy, octyl groups were able to effectively prevent the π-π stacking of conjugated chromophores and the highly emissive organic nanoparticles for cell imaging can be finally formed.

Figure 2. TEM images of (a) DTBT-0-, (b) DTBT-1-, and (c) DTBT-2-based FONPs under the optimized condition. 3.3 Molecular dynamics (MD) simulations

Molecular dynamics (MD) simulations were carried out to describe the mechanism underlying the SIEE strategy in aggregated nanoparticles. The simulations were conducted using the structures of DTBT-0, DTBT-1, and DTBT-2 in their single-state conformation, in THF solvent and water (Figure S7, Supporting Information). The optimized conjugated structure of DTBT-0 had relatively good co-planarity with a dihedral angle between benzothiadiazole and


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thiophene units of about 40°. After octyl chains were grafted onto DTBT-0, changes in conformation of the resulting DTBT-1 and DTBT-2 structures were negligible, which demonstrated that the conformation of chromophores were nearly unaffected by octyl chains or solvent. However, the distances of the three molecules in THF were different from those in water (Figure 3). Because these molecules could dissolve well in THF with the distances of over 2 nm (Insets, Figs. 3a-3c), their molecular interactions in THF could be ignored. Nonetheless, these molecules tended to aggregate in water due to their hydrophobic property (Insets, Figs. 3a-3c). Additionally, the probability of long molecular distance gradually increased with increasing numbers of octyl chains, and the molecular distances of all molecules were quite stable, especially in water (Figs. 3d-3f). It is known that fluorescence quenching is caused by strong molecular interactions, which induce electron and energy transfer among the neighboring molecules. Through the SIEE strategy, the interactions between chromophores were prevented by alkyl chains in aggregation state, and therefore, effectively reduced the π-π stacking while significantly improved the fluorescent properties.


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Figure 3. Left: Probability of distances distribution between two molecules in water and THF atmosphere (Inset: their corresponding schematic diagrams). Right: Distances between two molecules in water and THF atmosphere as functions of simulation time (a and d: DTBT-0, b and e: DTBT-1, c and f: DTBT-2). 3.4 Surface modification of DTBT-2 based FONPs and their optical properties

It is crucial for biomaterials to have good water dispersibility and biocompatibility.66 Herein, we modified the surface of DTBT-2-based nanoparticles with biomolecules of carboxylated-chitosan (CCHI), polyglycol (PEG), and polymine (PEI) for their impressive fluorescent properties. The Zeta potentials were then used to examine the modification (Table S3, Supporting Information). The Zeta potential of DTBT-2 nanoparticles in water was -19.9 mV,


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while that was changed to be -26.7, -74.1, and +36.4 mV after modifications with PEG, CCHI, and PEI, respectively. This indicated that the surface modification was successfully obtained here. As shown in Figure 4, mono-dispersed nanospheres with similar diameters of ~100 nm were observed for all these prepared nanoparticles.

Figure 4. TEM images of functionalized NPs: Non-functionalized FONPs (a), FONPs/PEG (b), FONPs/CCHI (c), and FONPs/PEI (d). Furthermore, water dispersibility of the surface-modified FONPs were significantly improved due to their enhanced electrostatic repulsions, which led to the highly stable FONPs (Figure 5 a). This is also one very important property for fluorescent nanoparticles in the application of cellular imaging. After that, photo-bleaching properties of DTBT-2 in thin film, non-functionalized FONPs, and surface-modified FONPs (including FONPs/PEG, FONPs/CCHI and FONPs/PEI) dispersed in water were also fully compared here. Although similar fluorescent spectra were observed for all nanoparticles (Figure S8 and S9, Supporting Information), photostability of FONPs were significantly improved and there were less photo-bleaching after long period of irradiation (Figure 5 b). This demonstrated that the DTBT-2-based FONPs could be effectively applied to use as fluorescent imaging agents.


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Figure 5. Stability of DTBT-2-based FONPs dispersed in water (a); Photo-bleaching properties of DTBT-2 in thin films and modified FONPs (b). 3.5 Cell viability and cell imaging

Figure 6. Cytotoxicity by WST-1 assay of HeLa and H9C2 cells treated with non-functionalized FONPs (a), FONPs/CCHI (b), FONPs/PEG (c) and FONPs/PEI (d).


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Figure 7. Confocal laser scanning microscopic images of HeLa cells treated with: (a, e, i) FONPs, (b, f, j) FONPs/CCHI, (e, g, k) FONPs/PEG and (d, h, l) FONPs/PEI, each at a concentration of 0.2 µg/mL for 30 min. (a-d): bright field images, (e-h): fluorescent images, and (i-l): merged images. WST-1 cell proliferation and cytotoxicity assay kit were used to investigate the cytotoxicity of DTBT-2-based FONPs. HeLa and H9C2 cells were treated with DTBT-2-based FONPs for 24 h. The results showed that over 90% cell viabilities were observed for the non-functionalized FONPs, FONPs/CCHI, and FONPs/PEG at a concentration of 100 µg·mL-1 (Figure 6), indicating that they have low cytotoxicity, but high biocompatibility.67 In contrast, the PEI-modified FONPs at higher concentrations of 50-100 µg·mL-1 showed noticeable toxicity to both HeLa and H9C2 cells due to its electropositive nature to the cell membrane.68 Then the HeLa and H9C2 cells were stained with our established FONPs, and their fluorescent confocal images were


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analyzed. As shown, all FONPs (FONPs/CCHI, FONPs/PEG, and FONPs/PEI) were able to stain both HeLa and H2C9 cells (Figure 7 and Figure S10, Supporting Information). Interestingly, these nanoparticles were differently localized in the cells. While nonfunctionalized FONPs localized in and/or around the cell membranes due to their low zeta potential, the modified FONPs with higher zeta potentials localized inside the cells. These findings indicate that the nanoparticles constructed using SIEE strategy not only have excellent fluorescence, but can also be used in bio-imaging (Figure S11, Supporting Information). The nanoparticles may also be used to stain various cellular organelles simply by adjusting their surface properties.

4. Conclusion In summary, a facile SIEE strategy, in which soft alkyl chains were introduced into chromophores, was used in the preparation of highly emissive FONPs. To prove the concept, octyl groups were grafted onto the fluorescent backbone of DTBT-0, forming two molecules of DTBT-1 and DTBT-2. Compared with DTBT-0, the markedly enhanced fluorescence was obtained from DTBT-1 and DTBT-2 in both the aggregated thin film and nanoparticles. Then the MD simulations were able to describe the mechanism underlying the SIEE strategy in aggregated nanoparticles. After modifications with various biocompatible molecules, DTBT-2-based FONPs with good water dispersibility and biocompatibility, were successfully applied in cellular imaging.

ASSOCIATED CONTENT Supporting Information:


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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.xxxxxx. 1H NMR spectrum,

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C-NMR spectrum, DLS data, Cytotoxicity

of FONPs, and Confocal images (PDF).

AUTHOR INFORMATION Corresponding Author: E-mail: [email protected] (L.J. Meng) Author Contributions: ‡

D. Dang and X. Wang contributed equally.

Notes: The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank the financial supports from the National Natural Science Foundation of China (Grant Nos. 21674085 and 51603165) and the Program for New Century Excellent Talents in University (Grant No. NCET-13-0453).

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