Chlorotoxin-conjugated, PEGylated Gd₂O₃ nanoparticles as a glioma-specific magnetic resonance imaging contrast agent

Abstract

An enhanced contrast in magnetic resonance imaging (MRI) with high specificity and low toxicity is of great importance in the accurate diagnosis and delineation of gliomas for improved outcomes. In this study, a glioma-targeted contrast agent was designed and prepared by conjugating chlorotoxin (CTX) to poly(ethylene glycol) (PEG) coated Gd₂O₃ nanoparticles (CTX-PEG-Gd₂O₃ NPs). The r₁ value of CTX-PEG-Gd₂O₃ NPs was measured to be 8.41 mM⁻¹ s⁻¹, which is higher than that of commercially available Gd-DTPA (4.57 mM⁻¹ s⁻¹). The T₁ contrast enhancement with a prolonged retention period up to 24 h within the brain glioma due to CTX conjugation was demonstrated. Moreover, cell viability and histological analysis verified the low cytotoxicity and the good biocompatibility of CTX-PEG-Gd₂O₃ NPs. Therefore, our study nominates CTX-PEG-Gd₂O₃ NPs as a promising glioma-targeted T₁ contrast agent that allows more accurate diagnosis and delineation of brain gliomas.

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Introduction

Gliomas are the most common primary malignant brain tumors with appalling mortality and disability rates due to their pattern of rapid and infiltrative growth. An accurate diagnosis of gliomas is critical in improving outcomes. Magnetic resonance imaging (MRI) is the gold standard in visualizing brain tumors. Especially, gadolinium (Gd)-chelates (e.g., Gd-DPTA) positive contrast-enhanced (T₁) MRI is a non-invasive method for the preoperative diagnosis and intraoperative localization of gliomas.^1,2 However, the short blood circulation time and the lack of specificity, together with an increasing concern of induced nephrogenic systemic fibrosis caused by the leak of Gd from the chelates,³ hinder their prevalence in clinical MRI. Alternatively, Gd-based nanoparticles (NPs) are emerging as more advanced contrast agents than Gd-chelates because these NPs not only present a large number of Gd ions within a small volume but also provide advantages such as prolonged blood circulation time, decreased toxicity, and easiness for further surface manipulations.⁴ Various Gd-based NPs, such as gadolinium oxide (Gd₂O₃),^5–13 gadolinium fluoride (GdF₃),¹⁴ gadolinium carbonate (GdCO₃),¹⁵ and gadolinium fluoride sodium (NaGdF₄),^16–18 have recently been exploited as novel T₁ contrast agents. Among them, Gd₂O₃ NPs exhibit a higher longitudinal relaxivity (r₁) and a significant positive contrast enhancement. Moreover, Gd₂O₃ NPs have been tentatively applied in MRI of brain tumors. Lee's group synthesized ultrasmall Gd₂O₃ NPs and confirmed their enhanced contrast effects in T₁-weighted MRI of a rat brain tumor.⁶ Another study conducted by Le Duc et al. showed that ultrasmall Gd₂O₃ based hybrid NPs induced a positive contrast for MRI of brain tumor.⁷

Target specificity is another critical requirement for a successful diagnosis and makes contrast agent safer by reducing the dosage and minimizing the damage to normal tissues.^11,19,20 The general strategy to achieve the specificity is to modify contrast agents with targeting ligands that could selectively bind the receptors overexpressed on the surfaces of cancer cells, such as folic acid,²¹ peptides,²² and antibodies.²³ In the case of glioma targeting, chlorotoxin (CTX), a 36-amino acid peptide, appears an attractive candidate as it can bind to matrix metalloproteinase-2 that is specifically upregulated in gliomas and related cancers, but poorly expressed in brain and normal tissues.^24–27 Zhang et al. conjugated superparamagnetic iron oxide NPs (SPIONs) with CTX and demonstrated that CTX conjugation significantly enhanced the uptake of SPIONs by brain cancer cells.²⁸ In contrast, the glioma-targeted MRI contrast agents based on CTX-conjugated Gd₂O₃ NPs to simultaneously enhance the contrast effects and prolong the retention period has not been reported so far.

Herein, a CTX conjugated, poly(ethylene glycol) (PEG) coated Gd₂O₃ (CTX-PEG-Gd₂O₃) NPs as glioma-targeted contrast agents was developed (Fig. 1). Specifically, oleate-coated Gd₂O₃ NPs were first prepared by thermal decomposition of gadolinium acetylacetonate in an organic medium to assure the uniformity and crystallinity. Water-dispersible Gd₂O₃ NPs was then obtained by the exchange of oleate ligands with a carboxylic silane, N-(trimethoxysilypropyl) ethylene diamine triacetic acid, trisodium salt (TETT). The abundant carboxylic groups of attached TETT silane allow the coupling of hetero-bifunctional PEG₂₀₀₀ (NH₂-PEG₂₀₀₀-COOH) to form PEG coated Gd₂O₃ NPs. The biocompatible PEG layer further helps to stabilize Gd₂O₃ NPs, to enhance the blood circulation, and to improve the water dispersible.^10,11,29 More importantly, it simultaneously serves as a linker for the conjugation of CTX and a stealth coating to reduce protein adsorption and non-specific macrophage uptake, ultimately prolonging serum half-life in vivo. The CTX-PEG-Gd₂O₃ NPs as a potential targeted MRI contrast agent toward brain glioma was evaluated in vivo using a glioma-bearing mouse model. Additionally, the toxicity of CTX-PEG-Gd₂O₃ NPs was evaluated both in vitro and in vivo by the methyl thiazoly tetrazolium (MTT) assay and the hematoxylin and eosin (H&E) staining, respectively.

Fig. 1 Schematic illustration of the preparation of CTX-PEG-Gd₂O₃ NPs.

Experimental

Materials

Gadolinium acetylacetonate, oleylamine, benzyl ether, and oleic acid (OA, >90%) were purchased from Sigma-Aldrich (USA). Polyethylene glycol (HOOC-PEG₂₀₀₀-NH₂) and N-(3-dimethylaminopropyl-N′-ethylcarbodiimide hydrochloride (EDC) were received from JenKem Technology Co., Ltd (Beijing, China). CTX was provided from Peptide Institute, Inc. (Shanghai, China). TETT silane (45% in water) was supplied by Gelest Inc. (Tokyo, Japan). N-Hydroxysuccinimide (NHS) was a product from Acros Oganics (Geel, Belgium). Fetal bovine serum (FBS) and Dulbecco's modified eagle's medium (DMEM) were acquired from Gibco (Basel, Switzerland). 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kit was received from Amresco (USA).

Synthesis of oleate-capped Gd₂O₃ (OA-Gd₂O₃) NPs

Oleate-capped Gd₂O₃ NPs were synthesized by the thermal decomposition method according to a reported protocol with minor modifications.³⁰ Briefly, 1.6 g of oleic acid, 1.6 g of oleylamine and 2 mmol of Gd (oleate)₃ were added to 20 mL of benzyl ether, which was degassed and stirred under vacuum at 100 °C for 1 h to remove water and air. Then, the solution was heated up to 290 °C and maintained at this temperature for 2.5 h under a nitrogen flow. After cooling down to room temperature, the OA-Gd₂O₃ NPs were precipitated with 20 mL of ethanol, collected by centrifugation, washed three times with ethanol, and redispersed in 10 mL of hexane for further use.

Synthesis of TETT silane modified Gd₂O₃ (TETT-Gd₂O₃) NPs

The water-dispersible TETT-Gd₂O₃ NPs were prepared by exchanging oleate ligands with TETT silane.^18,31 In brief, 100 mg of oleate-capped Gd₂O₃ NPs and 60 µL of acetic acid were added to 60 mL of toluene, followed by sonicating for 30 min. Then, 1.2 mL of TETT silane was added and the suspension was kept stirring at 70 °C for 48 h, during which precipitation occurred. The precipitated TETT-Gd₂O₃ NPs were collected and purified by sequentially washing three times with toluene and methanol, respectively. The precipitates were then dispersed in water and dialyzed against deionized water for 24 h using a cellulose dialysis bag (MWCO = 1000 Da). The TETT-Gd₂O₃ NPs were obtained by lyophilization.

Synthesis of PEGylated TETT-Gd₂O₃ (PEG-TETT-Gd₂O₃) NPs

To 10 mL of deionized water, 100 mg of TETT-Gd₂O₃ NPs was added. For activation of carboxylic groups, 29 mg of NHS and 23 mg of EDC were added. Then, 500 mg of bifunctional PEG (NH₂-PEG₂₀₀₀-COOH) was added to react with TETT-Gd₂O₃ NPs at pH value of 8.0 for 24 h. Then, the excess reactants were removed by dialysis against deionized water in a cellulose dialysis bag (MWCO = 3500 Da). The PEG-TETT-Gd₂O₃ NPs were obtained by lyophilization.

Synthesis of CTX-conjugated PEG-TETT-Gd₂O₃ (CTX-PEG-Gd₂O₃) NPs

100 mg of PEG-TETT-Gd₂O₃ NPs was added to 10 mL of deionized water, followed by the addition of 0.22 mg of NHS and 0.14 mg of EDC for activation. Then, 4 mg of CTX was added to solution and the pH value was adjusted to 8.0. The reaction was allowed to proceed under stirring at room temperature for 24 h. Then, the product was purified by dialysis against deionized water in a cellulose dialysis bag (MWCO = 3500 Da), followed by lyophilization to obtain CTX-PEG-Gd₂O₃ NPs.

Characterization

Transmission electron microscopy (TEM) images were obtained on a JEM-2100F (JEOL, Japan) microscope at an operating voltage of 100 kV. High-resolution transmission electron microscopy (HRTEM) image was acquired on a JEM-2100 (JEOL, Japan) electron microscope operated at 200 kV. X-ray diffraction (XRD) was performed on a Rigaku SmartLab X-ray diffractometer (Cu Kα radiation = 1.54056 Å) operating at 45 kV and 200 mA. The dynamic light scattering (DLS) and zeta potentials of the NPs were measured on a Nano-ZS90 Zetasizer (Malvern, UK). Fourier transform infrared (FTIR) spectra were recorded on a Nicolet Avatar 300 FT-IR spectrometer (Thermo Nicolet Instrument Corp., USA). A total of 100 scans were accumulated with a resolution of 4 cm⁻¹ for each spectrum. The content of Gd was determined on an inductively coupled plasma optical emission spectrometry (ICP-OES, Varian 710-ES, USA).

Relaxivity measurements

The PEG-TETT-Gd₂O₃ and CTX-TETT-Gd₂O₃ NPs were dispersed in the nanopure water and then diluted into desired Gd concentrations of 0, 0.0125, 0.025, 0.05, 0.1, and 0.2 mM. The T₁ measurements were performed on a small animal 7 T MRI instrument (Bruker Pharmascan 7T, Germany). The parameters were set as TR = 1500 ms and TE = 11 ms, 33 ms, 55 ms, 77 ms, and 99 ms, matrix size = 256 × 256, field of view (FOV) = 4.0 × 4.0 cm², flip angle (FA) = 180°, slice thickness = 1 mm. The corresponding relaxation time (T₁) was obtained for each dispersion and relaxivity values (r₁) were then calculated from the slope of the linear fitting of 1/T₁ relaxation time (s⁻¹) against Gd concentration (mM).

Cell culture

C6 glioma cells were cultured in Dulbeccos modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), streptomycin (100 µg mL⁻¹) and penicillin (100 U mL⁻¹) at 37 °C in a humidified 5% CO₂ atmosphere.

In vitro cytotoxicity assay

In vitro cytotoxicity was assayed by the MTT assay. C6 glioma cells were seeded in a 96-well plate at a density of 1 × 10⁵ per well. After 24 h of incubation, the medium was replaced by the suspension containing Gd at concentrations of 0, 1, 2.5, and 5 µg mL⁻¹ and incubated for another 24 h. Then, 100 µL of MTT (0.5 mg mL⁻¹) was added to each well and the plate was incubated for 4 h. The medium was removed and 150 µL of dimethyl sulfoxide (DMSO) was added to each well. Finally, the absorbance at 570 nm was measured using a Multiskan Spectrum microplate reader (Thermo Electron Corporation, USA) and the cell viability was calculated as a percentage compared to the control.

Histology

The ICR mice were sacrificed 21 days after injection of the TETT-Gd₂O₃, PEG-TETT-Gd₂O₃ and CTX-PEG-Gd₂O₃ NPs at a dosage of 6 mg Gd kg⁻¹. The major organs (heart, liver, kidney, spleen, lung and brain) were fixed in freshly prepared 10% formalin at 4 °C for 72 h. Then, the tissues were dehydrated with a tissue processor, embedded with paraffin blocks, sectioned into 2 µm slices, and stained with hematoxylin and eosin according to standard clinical pathology protocols. The stained sections were examined under a microscope (OLYMPU-BX53, Olympus Inc., Japan).

Animal model

The BALB/C-nude mice (male, 6–8 weeks old) were purchased from the academy of military medical sciences (Beijing, China) and were treated according to protocols evaluated and approved by the ethical committee of Capital Medical University. The glioma model was established according to a reported protocol.³² MRI on mice bearing brain gliomas was performed after tumor growing for 2 weeks.

In vivo MR imaging

The glioma-bearing mice were anesthetized by intraperitoneal injection of 6% chloral hydrate (0.10 mL/20 g body). In vivo T₁-weighted MR images were acquired before and after the injection of contrast agent (PEG-TETT-Gd₂O₃ or CTX-PEG-Gd₂O₃ NPs) at a dosage of 6 mg Gd kg⁻¹ by tail vein with a wrist coil on a small animal 7 T MRI scanner (Bruker Pharmascan, Germany). The measurement parameters were as follows, TR = 250 ms, TE = 10.0 ms, Flip angle = 180°, matrix = 256 × 256, FOV = 3.0 × 3.0 cm², slice thickness = 1.0 mm.

Results and discussion

To ensure good crystallinity, Gd₂O₃ NPs capped with oleic acid (OA-Gd₂O₃) were synthesized by thermal decomposition of gadolinium acetylacetonate at 290 °C in benzyl ether. The XRD pattern of OA-Gd₂O₃ NPs (calcined at 700 °C for 3 h) revealed five diffraction peaks (Fig. 2A), which could be readily indexed to the crystal phase of cubic Gd₂O₃ according to the Joint Committee for Powder Diffraction Standards (JCPDS) card no. 11-0604. A representative HRTEM image showed that a freshly synthesized OA-Gd₂O₃ NPs was nearly spherical in morphology (Fig. 2B) with an average size of 3.46 ± 0.74 nm (Fig. S1†). Note that the longitudinal relaxation of Gd₂O₃ NPs in such size range is significantly higher than those of NPs smaller or larger than this size.³³ Additionally, the lattice fringes observed in the HRTEM image further supported the good crystallinity of OA-Gd₂O₃ NPs (Fig. 2C). The distance between lattice fringes was measured to be 0.33 nm, corresponding to the (222) lattice planes of cubic phase Gd₂O₃.³⁴ The highly water-dispersible, glioma-targeted Gd₂O₃ NPs were then fabricated by replacing the OA ligands with the carboxylic silane (TETT), followed by sequential conjugation of heterobifunctional NH₂-PEG₂₀₀₀-COOH and CTX via a common EDC/NHS coupling technique to yield PEG-TETT-Gd₂O₃ and CTX-PEG-Gd₂O₃ NPs, respectively. The TEM images (Fig. 2D–F) revealed that these Gd₂O₃ NPs were well separated but with increased sizes (Fig. S1†). This is most likely due to the polymerization of TETT silane. Nevertheless, the as-prepared TETT-Gd₂O₃, PEG-TETT-Gd₂O₃, and CTX-PEG-TETT-Gd₂O₃ NPs could remain stable in PBS for weeks (insets, Fig. S1†). Moreover, the crystallinity of Gd₂O₃ NPs remained unchanged upon modifications (Fig. S2†).

Fig. 2 XRD pattern (A) and HRTEM images (B, C) of OA-Gd₂O₃ NPs. TEM images of (D) TETT-Gd₂O₃, (E) PEG-TETT-Gd₂O₃, and (F) CTX-PEG-Gd₂O₃ NPs. scale bar: B and C is 2 nm, D, E and F is 20 nm.

The composition and change of surface functional groups were characterized by FTIR spectroscopy. As shown in Fig. 3A, the IR spectrum of OA-Gd₂O₃ NPs showed the intensive bands at 2926 and 2855 cm⁻¹ attributed to the asymmetric and symmetric C–H stretching mode of –CH₂- groups. In addition, bands located at 1564 and 1450 cm⁻¹ were assigned to the asymmetric and symmetric stretching vibrations of COO⁻ groups.^18,35 Upon exchange of OA ligands with carboxylic silane TETT, these OA bands almost disappeared, whereas new characteristic bands of TETT such as the carboxylic groups at 1620 cm⁻¹, the Si–O–Si bond at 1086 cm⁻¹, and the N–C bond at 918 cm⁻¹ appeared (Fig. 3B).^13,18,35 The grafting of PEG onto the TETT-Gd₂O₃ was evidenced by the presence of PEG associated IR bands, such as –CH₂– peaks at 2886 cm⁻¹, –C=O bands at 1626 cm⁻¹, and C–O–C band at 1111 cm⁻¹ (Fig. 3C).¹¹ The conjugation of CTX led to the broadening and slight shift of IR band from 1626 to 1635 cm⁻¹ (Fig. 3D) due to the presence of both primary and secondary amines in residues such as lysine and arginine.²⁸

Fig. 3 IR spectra of (A) OA-Gd₂O₃, (B) TETT-Gd₂O₃,(C) PEG-TETT-Gd₂O₃, and (D) CTX-PEG-Gd₂O₃ NPs.

Furthermore, TGA and corresponding differential thermogravimetry (DTG) curves of NPs were obtained (Fig. 4). Weight loss observed from the DTG curves below 150 °C was caused by the removal of the absorbed water. At the temperature range of 200–550 °C, the DTG plot of OA-Gd₂O₃ NPs (Fig. 4A) displayed two major peaks, which could be ascribed to the thermal decomposition of initial and the outer layer of OA ligands.^36,37 Upon the ligand exchange of OA with TETT, the weight loss at the temperature range of 200–550 °C (Fig. 4B) due to TETT was obviously different from that of OA, suggesting that OA ligands were replaced by TETT. Similarly, the decomposition of PEG was found in the range of 300 to 450 °C for PEG-TETT-Gd₂O₃ NPs (Fig. 4C). The conjugation of CTX led to a slight increase in the weight loss (from 86.0% to 87.8%) and it should be noted that this additional weight loss occurred above 400 °C (Fig. 4D). This together with the IR analysis thereby confirmed the conjugation of CTX onto Gd₂O₃ NPs.

Fig. 4 TGA (black line) and DTG (blue line) curves of (A) OA-Gd₂O₃, (B) TETT-Gd₂O₃, (C) PEG-TETT-Gd₂O₃, and (D) CTX-PEG-Gd₂O₃ NPs.

Prior to the MRI study, in vitro cytotoxicity of Gd₂O₃ NPs toward C6 cells was evaluated by the MTT assay. Fig. 5 shows the C6 cell viability after incubation with different Gd₂O₃ NPs at equivalent Gd concentrations of 1, 2.5, and 5 µg mL⁻¹ for 24 h. As can be seen, the cytotoxicity of TETT-Gd₂O₃ NPs was concentration dependant. Nevertheless, the cell viability was still greater than 87% upon incubation with TETT-Gd₂O₃ NPs at the highest Gd concentration. As expected, PEGylated Gd₂O₃ NPs exhibited a declined cytotoxicity. Conjugation of CTX, however, led to an increased cytotoxicity, reflecting the enhanced cell uptake of Gd₂O₃ NPs due to the specific targeting ability of CTX. Overall, the MTT results confirmed the low cytotoxicity of these Gd₂O₃ NPs based MRI probes.

Fig. 5 Viability of C6 glioma cells after 24 h incubation with NPs (TETT-Gd₂O₃, PEG-TETT-Gd₂O₃ and CTX-PEG-Gd₂O₃) at various Gd concentrations. Each data represents the mean ± S.D. of three experiments.

Additionally, in vivo toxicity was investigated by histological analysis using the hematoxylin and eosin staining. Mice were sacrificed 21 day after injection of PBS (control), TETT-Gd₂O₃, PEG-TETT-Gd₂O₃, or CTX-PEG-Gd₂O₃ NPs at a dosage of 6 mg Gd kg⁻¹. Representative photographs of major organs such as brain, heart, kidney, spleen, liver, and lung are presented in Fig. 6. It was found that the structures of organs were normal and no apparent tissue damage, inflammation, or lesions were observed, suggesting the biocompatibility of Gd₂O₃ NPs. Nevertheless, long-term biocompatibility investigations are still required.

Fig. 6 H& E stained tissue sections from mice receiving no injection (control) and mice injected with TETT-Gd₂O₃, PEG-TETT-Gd₂O₃, or CTX-PEG-Gd₂O₃ NPs. Tissues were harvested from heart, liver, spleen, lung, brain and kidney 21 days post-injection.

The performance of Gd₂O₃ NPs (with or without CTX) as a contrast agent was evaluated on a 7T MRI scanner. The Gd contents of CTX-PEG-Gd₂O₃ and PEG-TETT-Gd₂O₃ NPs as determined by ICP-OES are 3.8% and 4% (w/w), respectively. The longitudinal relaxivity r₁ was then determined from the slope of the linear fitting of 1/T₁ (s⁻¹) vs. Gd concentration (mM). As illustrated in Fig. 7A, the r₁ values of CTX-PEG-Gd₂O₃ and PEG-TETT-Gd₂O₃ NPs were 8.41 and 8.06 mM⁻¹ s⁻¹, respectively. Both values are higher than that of Gd-DTPA and comparable to that of PEGylated Gd₂O₃ NPs reported in literature.^8–13 The high r₁ value could be attributed to the large number of Gd ions presented on the surface as well as the excellent water dispersible of NPs, which increase the contact of Gd ions to water and consequently accelerate the longitudinal relaxation of the water proton.⁶ Accordingly, the R₁ map images of both Gd₂O₃ NPs showed obvious contrast enhancement as the Gd concentration increased, suggesting the potential of Gd₂O₃ NPs as an efficient T₁ contrast agent.

Fig. 7 (A) r₁ measurement and (B) R₁ map images of Gd-DTPA, PEG-TETT-Gd₂O₃, and CTX-PEG-Gd₂O₃ NPs.

Next, the feasibility of CTX-PEG-Gd₂O₃ NPs as a targeted T₁ contrast agent toward glioma was investigated. Fig. 8 shows T₁-weighted MR images of glioma-bearing mice before and 15 min, 60 min and 24 h after intravenous administration of PEG-TETT-Gd₂O₃ or CTX-PEG-Gd₂O₃ NPs with an equivalent Gd dosage of 6 mg Gd kg⁻¹. It was found that PEG-TETT-Gd₂O₃ NPs slightly enhanced the contrast of the glioma region compared with the pre-contrast image. This is probably due to the fact that the dosage (6 mg Gd kg⁻¹) used in this study was less than that reported in literature.^8,9 Nevertheless, when glioma-bearing mouse was treated with CTX-PEG-Gd₂O₃ NPs at the equivalent dosage, a notable positive contrast enhancement was evidenced in the glioma region 15 min and 2 h post-injection, Moreover, apparent contrast between glioma and normal tissue remained 24 h post-injection. This was further evidenced by the quantitatively analysis of the contrast enhancement in brain gliomas (Fig. S3†),³⁸ which undoubtedly signifies the role of CTX conjugation. In addition, the preferentially accumulate of NPs in tumor over normal tissue regions led to a defined margin. This together with the improved contrast enhancement over a prolonged imaging period is particular valuable for intraoperative visualization of gliomas or monitoring of treatment response.

Fig. 8 MR images of the brains of C6 glioma bearing mice before and 15 min, 60 min and 24 h after intravenous injection of PEG-TETT-Gd₂O₃ and CTX-PEG-Gd₂O₃ NPs, respectively.

Conclusions

In summary, CTX-conjugated Gd₂O₃ NPs as a glioma-targeted contrast agent was prepared and characterized. The T₁-weighted MR images of the brain glioma-bearing mouse verified that CTX-PEG-Gd₂O₃ NPs could significantly enhance the glioma-tissue contrast and clearly delineate the glioma margin at a reduced dosage. This in line with the good biocompatibility makes CTX-PEG-Gd₂O₃ NPs an effective glioma-specific contrast agent for better preoperative diagnosis and intraoperative delineation of gliomas.

Acknowledgements

The authors gratefully acknowledge the financial supports from Natural Science Foundation of China (81271639, 81272805), Scientific Research Common Program of Beijing Municipal Commission of Education (KM20110025007), Beijing Municipal Foundation for the Talents (2011D005018000001), and the Basic-clinical Key Research Grant (13JL02) from Capital Medical University.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra10934a

‡ These authors contributed equally to this work.

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