Journal of Nanoparticle Research

, Volume 13, Issue 6, pp 2577–2585

Evaluation of the impact of chitosan/DNA nanoparticles on the differentiation of human naive CD4+ T cells

Authors

  • Lanxia Liu
    • Lab of Bioengineering, Institute of Biomedical EngineeringChinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials
  • Yuanyuan Bai
    • Lab of Bioengineering, Institute of Biomedical EngineeringChinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials
  • Dunwan Zhu
    • Lab of Bioengineering, Institute of Biomedical EngineeringChinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials
  • Liping Song
    • Lab of Bioengineering, Institute of Biomedical EngineeringChinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials
  • Hai Wang
    • Lab of Bioengineering, Institute of Biomedical EngineeringChinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials
  • Xia Dong
    • Lab of Bioengineering, Institute of Biomedical EngineeringChinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials
  • Hailing Zhang
    • Lab of Bioengineering, Institute of Biomedical EngineeringChinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials
    • Lab of Bioengineering, Institute of Biomedical EngineeringChinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials
Research Paper

DOI: 10.1007/s11051-010-0150-9

Cite this article as:
Liu, L., Bai, Y., Zhu, D. et al. J Nanopart Res (2011) 13: 2577. doi:10.1007/s11051-010-0150-9
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Abstract

Chitosan (CS) is one of the most widely studied polymers in non-viral gene delivery since it is a cationic polysaccharide that forms nanoparticles with DNA and hence protects the DNA against digestion by DNase. However, the impact of CS/DNA nanoparticle on the immune system still remains poorly understood. Previous investigations did not found CS/DNA nanoparticles had any significant impact on the function of human and murine macrophages. To date, little is known about the interaction between CS/DNA nanoparticles and naive CD4+ T cells. This study was designed to investigate whether CS/DNA nanoparticles affect the initial differentiation direction of human naive CD4+ T cells. The indirect impact of CS/DNA nanoparticles on naive CD4+ T cell differentiation was investigated by incubating the nanoparticles with human macrophage THP-1 cells in one chamber of a transwell co-incubation system, with the enriched human naive CD4+ T cells being placed in the other chamber of the transwell. The nanoparticles were also co-incubated with the naive CD4+ T cells to explore their direct impact on naive CD4+ T cell differentiation by measuring the release of IL-4 and IFN-γ from the cells. It was demonstrated that CS/DNA nanoparticles induced slightly elevated production of IL-12 by THP-1 cells, possibly owing to the presence of CpG motifs in the plasmid. However, this macrophage stimulating activity was much less significant as compared with lipopolysaccharide and did not impact on the differentiation of the naive CD4+ T cells. It was also demonstrated that, when directly exposed to the naive CD4+ T cells, the nanoparticles induced neither the activation of the naive CD4+ T cells in the absence of recombinant cytokines (recombinant human IL-4 or IFN-γ) that induce naive CD4+ T cell polarization, nor any changes in the differentiation direction of naive CD4+ T cells in the presence of the corresponding cytokines.

Keywords

ChitosanNanoparticleGene deliveryNaive CD4+ T cellNanomedicine

Introduction

The primary goals of developing nano-scaled carriers for drug delivery include more specific drug targeting and delivery, reduction in toxicity while maintaining the therapeutic effects, greater safety, and biocompatibility, as well as faster development of new medicines (De Jong 2008). Recently, polymeric nanoparticles have been widely investigated as carriers for drug delivery and adjuvant for vaccination. When employed as adjuvant or carriers for vaccine delivery, the nanomaterials are designed to target and stimulate the immune system for desired immune response, whereas under some circumstances, they entail avoiding interactions with the immune system to enhance the efficacy of drug/gene delivery and attenuate the adverse effects. It has been demonstrated that some forms of nanoparticles were able to suppress immune responses.

Polyamidoamine (PAMAM) dendrimers conjugated to glucosamine were reported to strongly inhibit induction of inflammatory cytokines and chemokines in human macrophages and dendritic cells (DCs) exposed to bacterial endotoxin (Shaunak et al. 2004). Fullerene derivatives could scavenge free radicals like superoxide dismutase and hence quenched the production of nitric oxide by macrophages (Chen et al. 2004). Cholesteryl butyrate-conjugated solid lipid nanoparticles were shown to reduce neutrophil adhesion to inflammation-activated endothelial cells (Dianzani et al. 2006). Poly(d, l-lactic/glycolic acid) (PLGA) nanoparticles functionalized with betamethasone reduced inflammation in experimentally induced arthritis in rats (Higaki et al. 2005). However, there were also reports showing that nanoparticles stimulated immune responses. It was found that glycoprotein, when immunized with lipid-coated polysaccharide nanoparticles, induced stronger immune response than when immunized with alum adjuvant (Castignolles et al. 1996). Small molecules conjugated to the surface of colloidal gold particles generated higher levels of specific antibodies than immunization of the same antigens with classical adjuvant (Dykman et al. 2004).

Chitosan (CS), a deacetylated product of chitin, has found many applications as a biomaterial in pharmaceutical and medical fields, owing to its excellent biocompatibility, biodegradability, and bioactivity. It is one of the most widely studied polymers in non-viral gene delivery since it is a cationic polysaccharide that forms nanoparticles with DNA and hence protects the DNA against digestion by DNase (Bloomfield 1996; MacLaughlin et al. 1998; Richardson et al. 1999; Mao et al. 2001). However, the impact of CS/DNA nanoparticle on the immune system still remains poorly understood. Previous investigations conducted by Chellat et al. (2005) and our lab did not find CS/DNA nanoparticles had any significant impact on the activity of human and murine macrophages that function both in the innate immune system through phagocytosis and production of cytokines, and in the adaptive immune system as a antigen presenting cell (Liu et al. 2009). To date, little is known about the interaction between CS/DNA nanoparticles and naive CD4+ T cells.

The T cells are divided into two types: CD4+ T and CD8+ T cells. CD8+ T cells are cytotoxic T cells which express the CD8 surface glycoprotein. CD4+ T cells, also termed T helper (Th) cells, express the CD4 surface glycoprotein and help other T cells in directing B- and T-cell responses. Th cells are differentiated from naive CD4+ T cells and are further classified into two major subsets that are commonly defined by the cytokines they secrete. Th1 cells secrete IFN-γ, inducing cellular immunity against intracellular pathogens. Th2 cells secrete IL-4 and mediate humoral immunity against extracellular pathogens (Zenewicz et al. 2009). This study was designed to investigate whether CS/DNA nanoparticles affect the initial differentiation direction of naive CD4+ cells. It was demonstrated that CS/DNA nanoparticles induced slightly elevated production of IL-12 by THP-1 cells, possibly owing to the presence of CpG motifs in the plasmid. However, this macrophage stimulating activity was much less significant as compared with lipopolysaccharide (LPS) and did not impact on the differentiation of naive CD4+ T cells. It was also demonstrated that, when directly incubating with naive CD4+ T cells, the nanoparticles induced neither the activation of the naive CD4+ T cells in the absence of recombinant human IL-4 (rhIL-4) or IFN-γ (rhIFN-γ) that induce naive CD4+ T cell polarization, nor any changes in the differentiation direction of the naive CD4+ T cells in the presence of the corresponding cytokines.

Experimental

Purification of plasmid DNA

The pGL3-control plasmid DNA (Promega Corp., USA) was purified using the QIAGEN Endofree Plasmid Mega Kits (Qiagen GmbH, Germany) according to the manufacturer’s instructions. The amount of LPS in the purified plasmid DNA was quantified by gel clot assay as described previously (Liu et al. 2009).

Preparation and characterization of CS/DNA nanoparticles

Chitosan/DNA nanoparticles (CGN) at a N/P ratio of 4:1 were prepared as described previously (Liu et al. 2009). Briefly, equal volume of plasmid DNA solution (0.02%) in 25 mM sterilized sodium sulfate solution and CS (MW: 50000–190000, degree of deacetylation: 75–85%, Sigma-Aldrich Corp., USA) solution (0.04%) in 0.2 M sterilized sodium acetate buffer (pH 5.5) were mixed together after preheating separately at 55 °C, and subsequently vortexed for 60 s. The resulting nanoparticles were characterized using a Brookhaven BI-9000AT digital autocorrelator at fixed scattering angle (θ) of 90° at room temperature. Each sample was measured three times.

Isolation of human naive CD4+ T cell

Peripheral blood mononuclear cells (PBMCs) were isolated from anticoagulated human blood by Ficoll density gradient centrifugation. The isolated PBMCs were suspended in PBS buffer containing 0.5% bovine serum albumin and 2 mM EDTA, and subsequently incubated with the biotin-antibody cocktails against CD8, CD14, CD16, CD19, CD36, CD45RO, CD56, CD123, TCRγ/δ, and Glycophorin A (Miltenyi Biotec, Germany) at 4–8 °C for 10 min, followed by incubating with anti-biotin microbeads for additional 15 min at 4–8 °C to label non-T helper cells and memory T cells. The cells were then washed by adding 2 mL of PBS/107 cells, centrifuged at 300×g for 10 min, and resuspended to 108 cells in 500 μL of PBS. The cell suspension was subsequently applied onto a LS column (Miltenyi Biotec, Germany) that was placed in the magnetic field of a MidiMACS separator (Miltenyi Biotec, Germany) and allowed to pass through the column. The effluent was collected as the fraction of unlabeled cells which represent the enriched naive CD4+ T cells. 106 enriched cells were stained with fluorochrome-conjugated antibodies against CD45RA (CD45RA-FITC) and CD4 (CD4-PE) (Miltenyi Biotec, Germany) followed by analyzing with flow cytometer (BD FACSCaliburTM, BD, USA).

ELISA for cytokine determination

The amount of IL-12, IL-4, and IFN-γ was determined using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. The test sensitivities of the ELISA Kits (RapidBio Lab, USA) employed for cytokine determination in the current study were 20 pg/mL for IL-12, 5 pg/mL for IL-4, and 10 pg/mL for IFN-γ. The kits were specific to the native cytokines released from the cells and do not recognize the recombinant cytokines. Briefly, 100 μL each of standard diluent and the test sample was added into a well of the 96-microtiter plate provided in the kits and incubated for 1 h at 37 °C. The incubation mixture was removed thoroughly and the wells were washed 5 times with washing buffer. 100 μL of biotin solution was subsequently added to each well and allowed for 1 h incubation at 37 °C. After washing, 100 μL of 1× HRP solution was added into each well and incubated for 30 min at 37 °C. Washing procedure was repeated and 50 μL each of color A and B solution was subsequently added into each well, followed by incubating for 9 min at 37 °C in the dark. After that, 100 μL of stop solution was added into each well and the absorbance at 450 nm was measured by a spectrophotometer (SPECTRAMax plus 384, MD, USA).

Indirect impact of nanoparticles on naive CD4+ T cells mediated by macrophages

In order to investigate whether CGN exposure would induce increased release of IL-12 by THP-1 human macrophages (ATCC, USA) and the indirect impact of CGN on the human naive CD4+ T cells that may be mediated by the IL-12 release, a transwell co-incubation system (Corning-Costar, USA) was employed, wherein a 0.1-μm diameter polycarbonate semi-permeable membrane lies between the lower and upper chamber. The enriched naive CD4+ T cells were adjusted to a density of 5 × 105 cells/mL and 0.5 mL of the cell suspension was seeded into the upper chamber of the transwell co-incubation system, with 0.7 mL of THP-1 cells being seeded into the lower chamber at a density of 1 × 105 cells/mL in DMEM supplemented with 10% FBS (Gibco, USA), 50 U/mL penicillin and 50 μg/mL streptomycin. CGN containing 5 μg/mL of DNA was co-incubated with the THP-1 cells in the lower chamber, using LPS (Sigma-Aldrich Corp., USA) at the concentration of 1 μg/mL as the positive control and the fresh culture medium alone as the negative control. The cell supernatants were collected at 12, 24, and 48 h after co-incubation at 37 °C in a humidified incubator with 5% CO2, aliquoted and stored at −80 °C for cytokine determination. The amount of IL-12, IL-4 and IFN-γ was determined by ELISA as described above.

Direct impact of nanoparticles on naive CD4+ T cells

The enriched naive CD4+ T cells were seeded into a 24-well plate at the density of 5 × 105 cells/mL. The cells were divided into six groups (6 wells/group) and incubated with CGN (containing 5 μg/mL of DNA), rhIL-4 (5 ng/mL) (R&D Systems, USA), rhIFN-γ (5 ng/mL) (R&D Systems, USA), CGN plus rhIL-4, CGN plus rhIFN-γ, and fresh culture medium alone, respectively. The cell supernatant was collected at 12, 24, and 48 h after co-incubation at 37 °C in a humidified incubator with 5% CO2, centrifuged, aliquoted, and stored at −80 °C for cytokine determination. The amount of IL-4 and IFN-γ was determined by ELISA as described above.

Statistical analysis

Data were presented as the mean of six individual observations with standard deviation. The statistical analysis was performed using the ANOVA (a one-way analysis of variance), followed by Bonferroni t-test for comparison with the control group. Statistical significance was determined at p < 0.05.

Results and discussion

It has been demonstrated that CS derivatives were effective immune adjuvants and CS microparticles were able to enhance both the systemic and local immune responses in mice (Seferian and Martinez 2000; Westerink et al. 2001; van der Lubben et al. 2003). It was also reported that CS could activate peritoneal macrophages and natural killer (NK) cells, enhance the production of antibodies and delayed-type hypersensitivity in guinea pigs (Nishimura et al. 1985). Marcinkiewicz et al. (1991) found that intraperitoneal administration of a water insoluble CS suspension enhanced humoral immune responses but not cellular immune responses in mice. Recently, several investigations have been conducted to investigate the feasibility of CS as a delivery cargo for DNA vaccines. Bivas-Benita et al. (2004) reported that pulmonary administration of DNA plasmid expressing different Mycobacterium tuberculosis epitopes in a formulation of CS/DNA nanoparticles could protect DNA from degradation by nucleases, induce DC maturation and result in increased IFN-γ secretion from T cells. Study conducted by Li et al. (2009) showed that oral administration of CS-encapsulated DNA expressing TGF-beta protein provided a long lasting TGF-beta1 expression in the intestine and a more durable alleviation of allergic inflammation in a mice model as compared to the administration of TGF-beta protein alone. However, neither of the investigations demonstrated the immuno-modulating effect attributed to CS. Seferian and Martinez (2000) found that CS particles, formulated in an emulsion with antigen, gave a prolonged, high antigen-specific antibody titer and sensitized animals for antigen-specific DTH responses, whereas CS particles alone offered no enhancement of an adaptive immune response. CS adipate was demonstrated to induce maturation and differentiation of thymocytes and regulates the number of specific cluster differentiation antigens on the surface of B splenocytes and lymph node T cells (Obminska-Mrukowicz et al. 2006).

As more investigations employ CS as a gene ferrying material, it is increasingly important to understand the immune responses to CS/DNA nanoparticles. Previous investigations conducted by Chellat et al. (2005) and our lab did not find CS/DNA nanoparticles had any significant impact on the activity of human and murine macrophages that function both in the innate immune response through phagocytosis and production of cytokines, and in the adaptive immune response as a antigen presenting cell. However, little is known about the impact of CS/DNA nanoparticles on T cells which play pivotal roles in the adaptive immunity. Naive CD4+ T cells can be differentiated in vitro to Th1 cells by culturing with IL-12 which is highly expressed by activated macrophages and dendritic cells (DCs). IL-12 activates STAT4 signaling pathways, resulting in elevated expression of cytokine IFN-γ, which in turn coordinates with IL-12 in promoting the differentiation of naive CD4+ T cells into Th1 cells (Watford et al. 2003). IL-4 stimulates the differentiation of naive CD4+ T cells toward the Th2 phenotype (Feili-Hariri et al. 2005). The primary goal of this study was to investigate the interaction of CGN with naive CD4+ T cells.

Contamination of the plasmid DNA with LPS may affect the assessment of the interaction between DNA nanoparticles and immune cells, since LPS was demonstrated to be a potent stimulator of cytokine production by immune cells (Koch et al. 2007; Mukherjee et al. 2009). Because of this, the QIAGEN Endofree Plasmid Mega Kits were employed to ensure the quality of the plasmid DNA. The purified plasmid DNA contained less than 0.1 EU/mL of LPS, which is usually regarded as endotoxin-free. As shown in Table 1, the CGN employed in the current study has an average diameter of 150.73 nm and a Zeta potential of 14.88 mV (Table 1).
Table 1

Characteristics of CGN

 

Mean size (nm)

Polydispersity index

Zeta potential (mv)

CGN

150.73 ± 3.20

0.199 ± 0.022

14.88 ± 0.79

In the current study, the human naive CD4+ T cells were isolated by depletion of non-helper T cells and memory CD4+ T cells. Naive CD4+ T cells can be distinguished from memory CD4+ T cells by the different expression pattern of CD45RA and CD45RO, both members of the CD45 family. CD45RA is expressed on naive CD4+ T cells, whereas CD45RO is expressed on memory CD4+ T cells. Therefore, the isolated naive CD4+ T cells were identified by staining with fluorochrome-conjugated antibodies against CD45RA and CD4 (Miltenyi Biotec, Germany) followed by analyzing with Fluorescence Activated Cell Sorter (FACS). It was demonstrated by FACS analysis that the purity of the enriched human naive CD4+ T cells which were double labeled with CD45RA-FITC and CD4-PE was 95.35%, as shown in Fig. 1.
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-010-0150-9/MediaObjects/11051_2010_150_Fig1_HTML.gif
Fig. 1

FACS analysis of the isolated naive CD4+ T cells stained with PE-conjugated anti-CD4 and FITC-conjugated anti-CD45RA

Nanoparticles might affect the differentiation of naive CD4+ T cells by direct mechanism or/and indirect mechanism which involves the mediation of IL-12 produced by other immune cells including macrophages. In order to investigate whether CGN exposure would induce increased secretion of IL-12 by THP-1 cells and the indirect impact of CGN on the human naive CD4+ T cells mediated by the IL-12 release, THP-1 cells and naive CD4+ T cells were incubated in a transwell co-incubation system and separated by a 0.1-μm diameter polycarbonate semi-permeable membrane which allows pass-through of macromolecules including cytokines. LPS is well known to interact with macrophages via Toll-like receptors (TLRs) and result in proinflammatory cytokines such as IFNγ, IL-2 (Gioannini et al. 2004; Pouliot et al. 2005) and IL-4 (Mukherjee et al. 2009), which in turn induce the differentiation of naive CD4+ T cells into Th1 or Th2 cells. Therefore, it was employed as an experimental control to ensure the reactivity of the THP-1 cells and naive CD4+ T cells to the inflammatory stimuli.

It was observed that direct exposure of CGN containing 5 μg/mL of DNA to THP-1 cells resulted slightly increased production of IL-12 by the macrophages (9.1 ± 1.7, 26.9 ± 4.3, and 65.4 ± 9.3 pg/mL, respectively, at 12, 24, and 48 h of exposure) as compared with the negative control wherein no detectable IL-12 was observed, whereas LPS induced a much higher IL-12 secretion (140.4 ± 15.7, 192.4 ± 5.7, 286.0 ± 16.2 pg/mL, respectively, at 12, 24, and 48 h of exposure) by THP-1 cells (Fig. 2a).
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-010-0150-9/MediaObjects/11051_2010_150_Fig2_HTML.gif
Fig. 2

Release of IL-12, IFN-γ, and IL-4 from the cells in the transwell co-incubation system. a IL-12, b IFN-γ, c IL-4. *p < 0.05 as compared with the negative control. https://static-content.springer.com/image/art%3A10.1007%2Fs11051-010-0150-9/MediaObjects/11051_2010_150_Figa_HTML.gif Detection limit

The amount of the native IFN-γ or IL-4 released from the cultured cells was employed as the indicator of the naive CD4+ T cell polarization since it was demonstrated that Th1 cells secrete IFN-γ and Th2 cells secrete IL-4. It should be noted that the ELISA kits employed in the current study only react with the native cytokines released from the cells and do not recognize the recombinant cytokines. Therefore, the cytokines detected herein are only the native cytokines released from the cells. There was no detectable release of IFN-γ or IL-4 observed in CGN exposure group within 48 h of exposure, whereas significantly elevated amount of IFN-γ (241.6 ± 14.6 pg/mL) and IL-4 (83.3 ± 9.0 pg/mL) was observed in the LPS exposure group at 48 h of exposure (Fig. 2b, c). These observations indicated that even though CGN induced slightly elevated production of IL-12 by THP-1 cells, this stimulating activity was much less significant as compared with LPS and did not impact on the differentiation of naive CD4+ T cells. Previous investigation in our lab found that the naked pGL3-control plasmid DNA employed in the current study was able to induce increased production of IL-12 by THP-1 cells, possibly owing to the presence of the CpG motifs in the plasmid DNA, which has been reported to induce maturation and activation of macrophages to produce cytokines including IL-12 (Cong et al. 2003; Kemp et al. 2003; Meng et al. 2003). However, this macrophage-stimulating activity was attenuated when the plasmid DNA was condensed by CS to form nanoparticles, possibly due to the binding of CpG motifs to CS, which led to decreased exposure of CpG motifs to THP-1 cells, implicating the absence of simulating activity of CS on macrophages.

The intent of investigating the direct impact of CGN on naive CD4+ T cells was two folds: to learn whether direct exposure to CGN would activate the polarization of naive CD4+ T cells, and whether exposure to CGN would impact on the differentiation of naive CD4+ T cells induced by the corresponding cytokines. The polarization of the naive CD4+ T cells directly exposed to CGN containing 5 μg/mL of DNA was investigated by observing the release of IL-4 or IFN-γ from the cells. No detectable production of IL-4 or IFN-γ was observed by the naive CD4+ T cells exposed to CGN, indicating the absence of activation of naive CD4+ T cells by CGN alone. To investigate the impact of nanoparticles on the polarization direction of naive CD4+ T cell induced by cytokines, the naive CD4+ T cells were exposed to the nanoparticles in the presence of hrIL-4 or hrIFN-γ, which was reported to induce the naive CD4+ T cells to differentiate into Th1 cells (IFN-γ) or Th2 cells (IL-4). There was no significant difference in the production of IL-4 by the naive CD4+ T cells exposed to CGN plus rhIL-4 or rhIL-4 alone and there was also no significant difference in the production of IFN-γ by the naive CD4+ T cells exposed to CGN plus IFN-γ or IFN-γ alone. These observations indicated that the presence of CGN did not affect the differentiation direction of naive CD4+ T cells induced by the corresponding cytokines (Fig. 3).
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-010-0150-9/MediaObjects/11051_2010_150_Fig3_HTML.gif
Fig. 3

Release of IL-4 and IFN-γ from the naive CD4+ T cells exposed to CGN, IL-4, IL-4+CGN, IFN-γ, IFN-γ+CGN, respectively. a IFN-γ released from the cells in contact with CGN or IL-4 or IL-4+CGN; b IFN-γ released from the cells in contact with CGN or IFN-γ or IFN-γ+CGN; c IL-4 released from the cells in contact with CGN or IL-4 or IL-4+CGN; d IL-4 released from the cells in contact with CGN or IFN-γ or IFN-γ+CGN. https://static-content.springer.com/image/art%3A10.1007%2Fs11051-010-0150-9/MediaObjects/11051_2010_150_Figb_HTML.gif Detection limit

Taken together, the current study did not find CS/DNA nanoparticles had any obvious impact on the differentiation of the naive CD4+ T cells in vitro, either directly or indirectly, adding more evidences to the fact that CS/DNA nanoparticles are immunologically safe. However, given the complex nature of the immune systems, further investigations on the interactions between CS/DNA nanoparticles and other immune cells are needed before a full conclusion could be drawn.

Acknowledgments

This research was jointly supported by the Ministry of Science and Technology of China (Grant No: 2005DIB1J094, 2006CB933203) and the National Natural Science Foundation of China (Grant No: 90406024).

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