Bacteria-Adsorbed Palygorskite Stabilizes the Quaternary

Apr 2, 2013 - Phosphonium Salt with Specific-Targeting Capability, Long-Term ... positive and Gram-negative bacteria. ... bacteria-adsorbed capability...
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Bacteria-Adsorbed Palygorskite Stabilizes the Quaternary Phosphonium Salt with Specific-Targeting Capability, Long-Term Antibacterial Activity, and Lower Cytotoxicity Xiang Cai,† Jinglin Zhang,† Yu Ouyang,† Dong Ma,‡ Shaozao Tan,*,† and Yilong Peng† †

Department of Chemistry and ‡Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China S Supporting Information *

ABSTRACT: In order to extend the antibacterial time of quaternary phosphonium salt in bacteria, palygorskite (PGS) is used as the carrier of dodecyl triphenyl phosphonium bromide (DTP), and a DTP-PGS hybrid is prepared. Antibacterial performance of this novel hybrid is investigated for both Grampositive and Gram-negative bacteria. The results show that the DTP could be absorbed on the surface of PGS which had bacteria-adsorbed capability. The DTP-PGS hybrid, combining the advantages of PGS and DTP, display specific-targeting capability, long-term antibacterial activity, and lower cytotoxicity, suggesting the great potential application as PGS-based antibacterial powder.

1. INTRODUCTION Clay minerals have attracted attention due to their nontoxic, environmentally friendly characteristic, and easy preparation by, e.g., their intercalation with selected organic1,2 or inorganic3,4 substances possessing antimicrobial properties, wide antimicrobial spectra, high security, and the synergistic effect with other antimicrobial materials.5,6 Palygorskite (PGS, the ideal formule is Si8O20(Mg,Al,Fe)5(OH)2(OH2)4·H2O), a widely used clay mineral, is 2:1 layer silicates. Unlike other clay minerals, the tetrahedral sheets of PGS are linked infinitely in two dimensions and have sheet-like and ribbon-like structure. With moderate surface charge, moderate cation exchange capacity (CEC), high specific surface area, and high adsorption capacity, the PGS was wildly used as drilling fluids, floor absorbents, cat litter, foundry sand binder, agricultural carriers, granulation binders, etc.7 Besides, because of their highly active surface, drugs such as hydrocortisone can be retained and subsequently released at an appropriate rate.8 Quaternary phosphonium salt (QPS) is a new generation of efficient, broad-spectrum organic antiseptic. With a number of advantages like low foam, strong capability of sludge stripping, and wide range of pH values, QPS has been extensively studied as active groups for preparing antibacterial materials.9,10 However, it is not safe to apply quaternary phosphonium salt directly in industrial and domestic applications because of antibiotic resistance in bacteria.2,11,12 Based on our previous work about antibacterial drug delivery carriers,13−16 in this paper, PGS was used as the carrier of dodecyl triphenyl phosphonium bromide (DTP, Figure S1, Supporting Information), and different amounts of QPS were © 2013 American Chemical Society

intercalated into PGS in aqueous solution to obtain a QPS− PGS hybrid. Then, the effects of QPS on the characteristics, morphology, release property, cytotoxicity and antibacterial mechanism of QPS−PGS were investigated, and the bacteriaadsorbed capability, long-term antibacterial effect, and lower cytotoxicity of this novel hybrid were demonstrated. The novelty of the present study was that the use of PGS as QPS carrier would make the use of QPS safer, and the specific benefits of this novel hybrid included (i) dose limitation to avoid eukaryotic toxicity, (ii) bacteria-adsorbed capability, (iii) dose control to achieve desired antibacterial effects, and (iv) specific-targeting capability.

2. EXPERIMENTAL SECTION 2.1. Materials. DTP of C.R. grade was supplied by Qingte Chemical Industry Co., Ltd. (Shanghai, China). Palygorskite (PGS) was purchased from Anji, Zhejiang province. Escherichia coli (E. coli) ATCC 25922 and Staphylococci aureus (S. aureus) ATCC 6538 were supplied by Guangdong Institute of Microbiology (Guangzhou, China). Luria−Bertani (LB) broth and nutrient agar culture medium were supplied by Huankai Microorganism Co., Ltd. (Guangzhou, China). Bovine serum was purchased from Aladdin Reagent Inc. (Shanghai, China). Thiazolyl blue tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Shanghai, China). Human nasopharyngeal carcinoma CNE1 (CNE1) cells were supplied by Xiangya School of Medicine, Central South University. All other reagents and solvents were obtained from commercial suppliers. All aqueous solutions were prepared with ultrapure water (>18 MΩ) from a MilliQ Plus system (Millipore). Received: March 18, 2013 Published: April 2, 2013 5279

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2.2. Preparation of Na-PGS and DTP-PGS. Original PGS was fully saturated to be Na-form using the ion exchange reaction with 1 M aqueous NaCl solution for 24 h at room temperature. Then, the turbid liquid was filtered and washed with deionized water until the reaction with 1% AgNO3 solution did not generate precipitation. After drying at 80 °C under vacuum for 1 day, the powder was gathered with a 300 mesh sieve (48 μm). And the resulting product was Na-PGS with cation exchange capacity of 65 mmol/100 g. The preparation of DTP-PGS was carried out by the following process: 10 g Na-PGS was dispersed in 490 g deionized water, to which the DTP was slowly added. The quantity of DTP was at various amounts (0.3 CEC, 0.5 CEC, 1.0 CEC, and 2.0 CEC) of Na-PGS to exchange sodium ions. Then, reaction mixtures were stirred vigorously at 65 °C for 6 h. The intercalated PGS was washed free from bromonium ions, and tested by 1% AgNO3 solution. After drying at 65 °C under vacuum, the intercalated PGS was gathered with 300 mesh sieve. The resulting intercalated clays were designated as DTP-PGS1, DTP-PGS2, DTP-PGS3, and DTP-PGS4, respectively. 2.3. Characterization. Fourier transform infrared spectrometer (FTIR) spectra of DTP-PGSs between 400 and 4000 cm−1 were obtained on a Nicolet 6700 spectrometer (USA). X-ray diffraction (XRD) patterns were recorded on a diffractometer (D/max-1200) using graphite monochromatic Cu Kα radiation (λ = 0.1541 nm) at a generator voltage of 40 kV and a current of 40 mA; measurements were conducted within a 2θ range of 2.0−30.0° at a scanning rate of 1°/min. Particle size and zeta potential were determined using a PALS Zeta Potential Analyzer (Brookhaven Instruments Co., USA) Thermogravimetric analysis (TGA) was conducted with a thermal analyzer (SDT-Q600). 10 mg sample was heated from 25 to 800 °C under N2 flow at a scanning rate of 10 °C/min. Transmission electron microscopy (TEM) was performed on Philips TECNAI-10 transmission electron microscope with an accelerating voltage of 200 kV. The samples were sliced with a microtome and the slices were placed in 200 mesh copper grids for analysis. 2.4. In Vitro Release Property. The release behavior of DTP from DTP-PGSs was performed, respectively, in pH = 7.4 phosphate buffered saline (PBS, the mixture of 5.55 g of Na2HPO4, 1.56 g of NaH2PO4, and 500 mL of deionized water) and bovine serum (used as received). Dialysis sack (Pore size: 12000 Da MWCO, Avg. flat width 35 mm, Sigma-Aldrich Co. LLC) was equilibrated with the dissolution medium for 3 h prior to experiments. After that, 100 mg DTP-PGSs in 20 mL PBS or bovine serum was taken in the dialysis sacks. Then, dialysis sack was dipped into receptor compartment containing 100 mL PBS or bovine serum, and then shaken at 37 ± 0.5 °C with 100 rpm shaking frequency. The receptor compartment was closed to prevent the evaporation losses from the dissolution medium. 5 mL of PBS or bovine serum was withdrawn at regular time intervals and the same volume was replaced with a fresh dissolution medium. The concentrations of DTP in the filtered liquid were measured using inductive coupled plasma emission spectrometer (ICP, TJA IRIS (HR)). The release studies were made in triplicate and the results were reported as average. 2.5. Minimum Inhibitory Concentration Tests. The minimum inhibitory concentration (MIC) of DTP-PGSs against E. coli and S. aureus was measured by 2-fold dilution method. Briefly, DTP-PGSs were suspended into Mueller-Hinton broth medium to form homogeneous suspensions and then 2-fold diluted into different concentrations. Each 1 mL of culture medium containing various concentrations of test sample was inoculated with 0.1 mL of 106 cfu/ mL bacterial suspensions and cultured for 24 h at 37 °C under shaking. Then the growth of bacteria was observed. When no growth of bacteria was observed in the lowest concentration of test sample, the MIC of the sample was defined as this value of dilution. The test for every MIC of DTP-PGSs was repeated three times. 2.6. Bacterial Growth Analysis. The blank control as well as DTP-PGSs was assayed for antibacterial activity using growth inhibition studies against Gram-negative bacteria E. coli strain. The E. coli growth in the culture media was monitored by measuring its optical density (O.D) at 600 nm. 10 μL of mid log phase (i.e., O.D. = 0.3 units) E. coli was inoculated in 100 mL of freshly prepared nutrient

LB broth medium supplemented with 200 mg/L of DTP-PGSs. Negative controls consisting of LB medium with only inoculum were used. All the flasks were then incubated at 37 °C in an orbital shaker at 150 rpm. The bacterial growth was monitored at an interval of 1 h by measuring the O.D. of the culture media at λ = 600 nm.17 2.7. Ion Concentration Analysis. After growing to primary-log phase at 37 °C, the E. coli was collected, washed, and diluted to 105− 106 cfu/mL with 0.8 wt % saline water. 200 mg/L of DTP-PGS3 was dispensed into 10 mL of a sterile 0.8 wt % saline water containing about 105−106 cfu/mL of E. coli. The solutions were then incubated at 37 °C for 6 h with continuous shaking, and 5 mL dispersions were drawn each hour, followed by being centrifuged at 5000 rpm for 2 min. The centrifugation suspension procedure was repeated 5 times to thoroughly remove the culture medium. Finally, 10 mL of 10 mol/L nitric acid was added into the supernate. The contents of Ca2+, K+, and Mg2+ in the solutions were measured by an inductively coupled plasma optical emission spectrometer (ICP, Optima 2000DV, America).18

3. RESULTS AND DISCUSSION 3.1. Structure and Properties of DTP-PGSs. The existence of DTP in DTP-PGSs was proved by FTIR (Figure S2, Supporting Information). XRD patterns of Na-PGS and DTP-PGSs were shown in Figure 1. It was denoted that the

Figure 1. XRD patterns of (a) Na-PGS, (b) DTP-PGS1, (c) DTPPGS2, (d) DTP-PGS3, (e) DTP-PGS4.

PGS used in this research was heterogeneous. The PGS was the one component phase, containing appreciable amount of quartz. The reflection at 2θ = 26.7° (d = 0.334 nm) was related to the quartz impurities. XRD pattern of Na-PGS displayed a diffraction peak at 2θ = 8.38°, which was assigned to the basal spacing (d011) of 1.05 nm. In XRD patterns of DTP-PGSs, the new diffraction peaks at 2θ = 2.88° were observed (d = 3.07 nm). The structure of PGS was shown in Figure 2a. The tetrahedral sheets are linked infinitely in two dimensions, and they are structurally different from other clay minerals in that the octahedral sheets are continuous in only one dimension and the tetrahedral sheets are divided into ribbons by the periodic inversion of rows of tetrahedrons. So, the chemical compounds “intercalated” into 5280

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Figure 2. Molecular structures of (a) PGS, (b) DTP, (c) DTP-PGS.

Table 1. Structure and Property Parameters for Na-PGS and DTP-PGSs samples

DTP contentsa(mass%)

Na-PGS DTP-PGS1 DTP-PGS2 DTP-PGS3 DTP-PGS4

 4.2 9.9 13.4 15.8

zeta potentialb(mW) −31.2 −27.7 −16.1 −12.5 −8.7

± ± ± ± ±

2.1 1.8 2.4 0.6 0.2

c

0.186 0.197 0.234 0.296 0.276

PDI ± ± ± ± ±

0.003 0.013 0.010 0.008 0.011

Z-averaged(d.nm) 1022 1438 1592 1732 1835

± ± ± ± ±

275 501 413 533 452

The weight loss between 200 and 500 °C (Figure S3, Supporting Information). b±SD, n = 3. c±SD, n = 3. PDI (polydispersity index) is size distributions. d±SD, n = 3. a

the PGS will not be “intercalated” into other clay minerals, i.e., montmorillonites. However, there are some channels in PGS, and the dimensions of the channels are approximately 4 Å by 6 Å.7,19 These channels are filled with what is termed zeolitic water, which means these channels are supposed to be hydrophilic. When this water is driven off and thus the sorptivity is increased, chemical compounds of smaller sizes that will fit into these channels are readily absorbed. Based on the bond lengths and bond angles, the length of a DTP is about 2.45 nm (Figure 2b), the size of hydrophilic end of DTP is 0.98 × 0.93 nm2 (Figure 2b), and the size of hydrophobicity end of DTP is 0.22 × 0.18 nm2 (Figure 2b). So, the hydrophilic end of DTP could not be absorbed into the channels (Figure 2c). In other words, the DTP could only be absorbed on the surface of PGS, and form loose DTP crystal (Figure 2c). The hypothesis was consistent with the XRD result. In addition, the loose PGS crystal was enhanced gradually with the increase of DTP content. 3.3. TGA Analysis. TG analysis was carried out to determine the chemical composition of as-prepared DTPPGSs. From the TG analysis (Figure S3, Supporting Information), the DTP contents could be determined in range (2) (200−400 °C), and they are shown in Table 1. 3.4. TEM Analysis. Figure 3 showed the TEM images of PGS, Na-PGS, and DTP-PGSs. Both PGS (Figure 3a) and NaPGS (Figure 3b) were elongate in shape and often occurred as bundles of elongate and lath-like particles. The bundles of the DTP-PGSs were agglomerated. With the increase of the DTP

Figure 3. TEM images of (a) PGS, (b) Na-PGS, (c) DTP-PGS1, (d) DTP-PGS2, (e) DTP-PGS3, (f) DTP-PGS4.

content, the bundles of the DTP-PGSs were more agglomerated, and tended to become dark. The dark, inner shadows were suggested to be DTP. Zeta potential and particle size of Na-PGS and DTP-PGSs in Table 1 also showed that the particle sizes of DTP-PGSs were 5281

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Figure 4. Release property of DTP-PGSs in (a) bovine serum and (b) PBS at 37 ± 0.5 °C with 100 rpm shaking frequency.

obviously increased due to the decrease of absolute value of zeta potential of organo-clay minerals. 3.5. Release Analysis. The release quantity of DTP from DTP-PGSs at different media was shown in Figure 4. The results showed that DTP on DTP-PGSs exhibited negligible release and 26−37% release from DTP-PGSs in PBS and bovine serum in 72 h, respectively. DTP was releasing continually in bovine serum with the lapse of soaking time, and the released quantity of DTP from DTP-PGS1, DTPPGS2, DTP-PGS3, and DTP-PGS4 was 17.3, 21.1, 26.4, and 31.3 wt % after 72 h, respectively. The slow but finite release of DTP in serum was likely caused by the binding between DTP and serum proteins, thus the DTP-PGSs had specific-targeting capability.13 3.6. MIC Analysis. Table 2 showed the MIC of PGS and DTP-PGSs. PGS showed poor antibacterial activity against E.

Figure 5. Growth patterns of E. coli treated with (a) blank control, (b) DTP-PGS1, (c) DTP-PGS2, (d) DTP-PGS3, and (e) DTP-PGS4 after 8 h in LB medium.

Table 2. MIC of PGS and DTP-PGSs MIC/mg·L−1 samples PGS DTP-PGS1 DTP-PGS2 DTP-PGS3 DTP-PGS4

E. coli

found to be enhanced with the increasing concentrations of DTP-PGSs treated in culture media. For DTP-PGS1, the growth curve of the E. coli was similar to that of the blank control. For DTP-PGS2 and DTP-PGS3, the growth curves of the E. coli were delayed in the onset of the logarithmic growth phase, referred to as bacteriostatic condition. Significant delay in the onset of the growth were observed for the E. coli treated with DTP-PGS2 (∼6 h) and DTP-PGS3 (∼5 h), and its corresponding inhibitions of growth were estimated to be ∼50% and ∼70% with respect to the control batch, respectively. For DTP-PGS4, complete inhibition of the bacterial growth was observed for the E. coli, corresponding to minimum bactericidal concentration. It might be surmised that the antibacterial effect of DTP-PGSs was dependent on the concentration of DTP treated in the culture media. This result was in good agreement with the previous reports.20,21 3.8. Ion Concentration Analysis. The selective permeability of the outer membrane barrier allows the normal cell to absorb the essential nutritions and to eliminate noxious substances.16 Owing to the increase of permeability of cell membrane with the treatment of DTP-PGS3, it was expected that the cell became more permeable to the milieu. Here, DTPPGS3 was added to the native and treated cell suspensions. The ICP-AES measurement of K+, Ca2+, and Mg2+ in Table 3 showed that there were much higher K+, Ca2+, and Mg2+ contents in the treated cell extracellular fluid, indicating that the selectivity in the absorption of the substance in the milieu dropped significantly.

S. aureus >10000 600 450 300 200

>10000 500 325 200 125

coli and S. aureus, because both of the MIC values were higher than 10 000 mg·L−1. For DTP-PGSs, they showed relatively high antibacterial activity against E. coli and S. aureus, and the antibacterial activity was enhanced along with the increase of the DTP content. In addition, DTP-PGSs exhibited lower activity against E. coli than against S. aureus. The structure of the cytoderm of E. coli is more complicated than that of the S. aureus because another layer outside of the peptidoglycan layer is called outer membrane, which is composed mainly of lipopolysaccharides and phospholipids. The outer membrane took a significant role to protect bacteria cells against foreign compounds such as DTP-PGSs. Thus, the lower sensitivity of DTP-PGSs toward E. coli was mainly due to the presence of the outer membrane.13−16 3.7. Growth Curve of E. coli. The growth patterns of E. coli treated with DTP-PGSs in 100 mL of LB media were assayed (Figure 5). For the blank control, the exponential phase appeared to last for about 8 h, and then the growth of E. coli entered stationary phase. The inhibition of bacterial growth was 5282

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bacterial cell walls;24,25 (3) drug loadings of modified clays;26,27 (4) particle size of clay minerals;28,29 and (5) the antimicrobial activity was the synergistic effect of the amount of drug released, surface charge, and particle size of organo-clay minerals.2 As is well-known, DTP is an organic antibacterial agent, and is dangerous to living organisms. Therefore, the mechanism of antibacterial activity of DTP-PGSs was suggested as follows: (1) Due to the bacteria-adsorbed capability of PGS,22 bacterial cells were trapped in DTP-PGSs; (2) the “elongate needle” of PGS of DTP-PGSs damaged the cytoplasmic membrane of the bacterial cell (Figure 6); (3) the DTP of DTP-PGSs was released and reacted with cytoplasmic constituents; (4) the normal physiologic activity of the cell was completely interrupted and the cell died. In other words, the elongate needle shape of PGS was in contrast to the flake-shaped graphene which led to some unique applications:13 the PGS was like an “elongate needle”, and the DTP-PGS acted as a “needle with poison” where the DTP was the “poison”. The DTP-PGSs combined the advantages of both PGS and DTP on antibacterial activity, rendering the specific-targeting capability of DTP (only release DTP in bacterial cells) and the safer and more efficient use of DTP. 3.10. Cytotoxicity Test. We also carried out cytotoxicity test on the DTP-PGSs. The MTT assays (Figure 7) showed

Table 3. Concentrations of K+, Ca2+, and Mg2+ in the 200 mg/L DTP-PGS3 Treated Cell Extracellular Fluid samples

time (h)

K+ (mg/L)

Ca2+ (mg/L)

Mg2+ (mg/L)

blank control

0 2 6 12 0 2 6 12

0 0.912 1.968 2.687 0 3.673 5.487 6.024

0 0.066 0.089 0.121 0 0.212 0.401 0.478

0 0.021 0.065 0.094 0 0.255 0.276 0.291

DTP-PGS3

3.9. Antibacterial Mechanism. Figure 6 showed the images of normal E. coli and S. aureus cells treated with PGS and DTP-PGS3, respectively. Outer membrane plays an important role in maintaining the morphology and protecting the cell.13 As shown in Figure 6, the normal E. coli (Figure 6a) and S. aureus (Figure 6e) cells demonstrated continuous smooth outer membrane. After 8 h treatment with PGS, the PGS was absorbed onto the E. coli (Figure 6b) and S. aureus (Figure 6f) cells. The outer membrane was not separated from the E. coli (Figure 6b) and S. aureus (Figure 6f) cells, suggesting that PGS had poor antibacterial activity against E. coli and S. aureus, which was coincide with the MIC test. The elongate PGS particles varied in length from about 1022 ± 275 nm (Table 1) and were approximately 50 nm (Figure 3 and Figure 6) in diameter. This shape and size resulted in high surface area of PGS, and could make PGS act as an adsorbent for bacteria, and even viruses.22 In our previous study, we also found the organo-clay mineral with the same particle-size-like bacterial size exhibited better bacteria-adsorbed capability.2 So, we suggested that PGS had bacterial adsorption capability. After 2 h treatment with DTP-PGS3, the outer membrane was partly separated from the E. coli (Figure 6c) and S. aureus (Figure 6g) cells. After 8 h treatment with DTP-PGS3, the outer membrane was separated from E. coli (Figure 6d) and S. aureus (Figure 6h) cells, and litter flocculent substances left from cell surface. In previous studies, antimicrobial effects by which several clay minerals acted were notably explained as follows: (1) interaction phenomena between organic molecules23 (e.g., humic acid), inorganic ions, and microorganisms; (2) the formation of C−O−Na−Si complexes on the surfaces of

Figure 7. Cytotoxicity of (a) DTP, (b) DTP-PGS4, (c) DTP-PGS3, (d) DTP-PGS2, (e) DTP-PGS1, and (f) PGS on CNE1 cells.

Figure 6. TEM images of normal E. coli cells (a) and S. aureus cells (e), E. coli cells (b) and S. aureus cells (f) treated by PGS after 8 h in LB medium, E. coli cells (c) and S. aureus cells (g) treated by DTP-PGS3 after 2 h in LB medium, E. coli cells (d) and S. aureus cells (h) treated by DTP-PGS3 after 8 h in LB medium. 5283

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that PGS (5 μg/mL) exhibited a slight cytotoxicity (∼5%) to CNE1 within 24 h incubation. PGS of higher concentration (500 μg/mL) led to increased cytotoxicity (∼30%) within 24 h. DTP exhibited serious cytotoxicity to CNE1 within 24 h incubation (the cell viability of CNE1 was reduced to 53% and 8% with DTP-PGSs of 5 and 100 μg/mL, respectively). However, the cell viability of CNE1 was reduced to 72−86% and 30−50% with DTP-PGSs of 5 and 500 μg/mL, respectively. Therefore, the cytotoxicity of DTP-PGSs was significantly lower than DTP, and the use of DTP-PGSs would be safer than the direct use of DTP, which was in accordance with the result of the inverted phase contrast microscope measurements (Figure S4). Such a difference in cytotoxicity might arise from the different surface charges of DTP and DTP-PGSs surfaces.13 In addition, the cytotoxicity of DTPPGSs increased with the increasing amount of DTP content. Such a difference in cytotoxicity might arise from the different release content of DTP (Figure 4). Compared with other reports,13−15 we concluded that DTP-PGSs were relatively biocompatible nanomaterials with slight cytotoxicity.

ASSOCIATED CONTENT

S Supporting Information *

Details of the structure of dodecyl triphenyl phosphonium bromide, the cytotoxicity assay, the statistical analysis, the FTIR spectra analysis, the TGA curves analysis, and the morphologic changes of CNE1 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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4. CONCLUSIONS In summary, PGS was used as the carrier of DTP, and a DTPPGS hybrid was prepared. The results showed that the DTP could be absorbed on the surface of PGS. The DTP-PGS hybrid exhibited negligible DTP release in PBS and 26−37% DTP release in serum over 72 h, rendering the DTP specifictargeting capability. The antibacterial activity of DTP-PGS was enhanced along with the increase of DTP content, and the antibacterial effect of DTP-PGSs was dependent on the concentration of DTP in the culture media. After treatment with DTP-PGS, the selectivity of substance absorption in the milieu dropped significantly. The “elongate needle” of PGS had bacteria-adsorbing capability, and the DTP-PGSs combined the advantages of both PGS and DTP on antibacterial activity. Thus, the use of DTP would be safer and more efficient. The use of PGS as an antimicrobial carrier would realize a variety of antibacterial applications of PGS.

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AUTHOR INFORMATION

Corresponding Author

*E-mail addresses: [email protected] (Shaozao Tan). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank the National Natural Science Foundation of China (21006038, 21271087, 51172099, 20971028, and 21176100), the Natural Science key Foundation of Guangdong Province of China (10251007002000000), Foundation of Science and Technology Projects of Guangdong Province (No. 2011B010700080), and the Fundamental Research Funds for the Central Universities (21612109). 5284

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