Facile synthesis of fluorescent Au/Ce nanoclusters for high-sensitive bioimaging
© Ge et al.; licensee BioMed Central. 2015
Received: 26 September 2014
Accepted: 23 January 2015
Published: 3 February 2015
Tumor-target fluorescence bioimaging is an important means of early diagnosis, metal nanoclusters have been used as an excellent fluorescent probe for marking tumor cells due to their targeted absorption. We have developed a new strategy for facile synthesis of Au/Ce nanoclusters (NCs) by doping trivalent cerium ion into seed crystal growth process of gold. Au/Ce NCs have bright fluorescence which could be used as fluorescent probe for bioimaging.
In this study, we synthesized fluorescent Au/Ce NCs through two-step hydrothermal reaction. The concentration range of 25–350 μM, Au/Ce NCs have no obvious cell cytotoxicity effect on HeLa, HepG2 and L02 cells. Furthermore, normal cells (L02) have no obvious absorption of Au/Ce NCs. Characterization of synthesized Au/Ce NCs was done by using TEM, EDS and XPS. Then these prepared Au/Ce NCs were applied for in vitro/in vivo tumor-target bioimaging due to its prolonged fluorescence lifetime and bright luminescence properties.
The glutathione stabilized Au/Ce NCs synthesized through hydrothermal reaction possess stable and bright fluorescence that can be readily utilized for high sensitive fluorescence probe. Our results suggest that Au/Ce NCs are useful candidate for in vitro/in vivo tumor bioimaging in potential clinical application.
Cancer is still a serious threat to human health and its effective treatment is still a big challenge. Its early diagnosis provides opportunity for the effective treatment and hence can improve the survival rate. The early diagnosis have been researched extensively through finding biomarkers [1,2] and in situ fluorescent bio-imaging [3,4]. Fluorescence imaging has been introduced as an important bioimaging tool and in vivo fluorescence imaging can improve the visibility of infected site.
Recently, many fluorescent composites have been developed as sensitive optical imaging probes, including fluorescent dyes [5,6], quantum dots [7-10], metal nanoparticles [11-13] and up-converted nanomaterials [14,15]. Precious metals like gold and silver nanomaterials have been paid much attention for application in a wide range of biomedical field due to their excellent biocompatibility and physicochemical properties [16-18]. Folic acid conjugated AuNCs@SiO2 nanoprobes (AuNCs@SiO2-FA) with good biocompatibility have been designed and applied into fluorescent imaging of gastric cancer cells . Meanwhile, various kinds of metal nanoclusters have been synthesized by coating biological ligands such as BSA, PEG, DHLA and GSH, etc., and accordingly, different kinds of biomedical function have been developed to meet the practical and clinical needs . In addition, lanthanide-doped luminescent nanomaterials have been also explored for some disease diagnosis [20,21]. Ce3+ and Eu3+ co-doped multifunctional nanoparticles like NaGdF4:Ce3+, Eu3+ NPs have been also explored for bioimaging . Moreover, some studies reported the combining of various detection mediums like quantum dots and magnetic nanoparticles for multi-mode imaging . In view these observations, a new strategy for facile synthesis of fluorescent Au/Ce nanoclusters (NCs) has been explored in this contribution by doping trivalent cerium ion into seed crystal growth process of gold. Through hydrothermal synthesis of these glutathione stabilized nanoclusters, it is possible to utilize the biocompatible and fluorescent Au/Ce NCs to realize high-sensitive in vitro/in vivo tumor-target bioimaging.
Results and discussion
Characterization of Au/Ce NCs and Au NCs
Several noble metal clusters have shown remarkably long fluorescence lifetimes of 1 or 2 orders of magnitude higher than organic dyes and quantum dots . As shown in Figure 4B, the fluorescence lifetime determined by time-correlated single photon counting indicates that Au/Ce NCs exhibit a luminescence lifetime with a biexponential decay; a short component τ1 (2.34 ± 0.05 μs, 87.2%) and a long component τ2 (11.25 ± 0.30 μs, 12.8%), respectively. The microsecond time scale long lifetime component maybe due to the electron transfer from the clusters to the ligand where a redox process might occur and charge-separated trap , hence, making it possible to avoid the interference of short-lived background fluorescence. The apparent long fluorescence lifetime of Au/Ce NCs makes it possible for their future bio-application in monitoring some important biological process through biosensing or bioimaging.
Application of Au/Ce NCs in bioimaging
In summary, a novel strategy of facile Au/Ce nanoclusters synthesis by doping trivalent cerium ion into seed crystal growth process of gold has been developed. The EDS, UV–Vis absorption spectroscopy, fluorescence and XPS characterization validates that the glutathione stabilized Au/Ce NCs via hydrothermal synthesis possess stable and bright fluorescence. The as-prepared GSH–Au/Ce NCs have strong fluorescence and long fluorescence lifetime, which could be readily utilized for high sensitive tumor-target bioimaging.
Chemicals, materials and cells
Auric chloride acid (HAuCl4 · 6H2O), L-glutathione (GSH) and Cerium nitrate (Ce(NO3)3 · 6H2O) along with all chemicals (analytical reagent, AR) were purchased from Sinopharm Chemical Reagent Co., Ltd. DMEM medium, fetal bovine serumwere (FBS) and penicillin were purchased from SunShineBio Technology Co., Ltd (Nanjing, China). Ultrapure water (18.2MU cm; Milli-Q, Millipore) was used as lyticagent of all aqueous solutions reagent. Tumor cells like HeLa cells, HepG2 cells and normal cells like L02 cells (purchased from Cell Bank of Chinese Academy of Sciences, Shanghai) were applied in our study. All of them were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin at 37°C in a carbon dioxide cell incubator with 5% CO2 and 95% relative humidity.
Transmission Electron Microscopy (TEM) images were recorded using a JEM-2100 microscope (JEOS, Japan) to characterize the size and size distribution, a certain concentration of sample solution was spotted on carbon coated copper grid (300 meshes) and was dried in desiccator at room temperature. Energy dispersive X-ray spectroscopy (EDS) analyses was done in a Zeiss Ultra Plus scanning electron microscopic (SEM). The valence state of gold and cerium atoms in the Au/Ce nanoclusters was investigated by a PHI 5000 VersaProbe X-ray photoelectron spectrometer (XPS), briefly, samples were droped on a silicon wafer and dried in laboratory ambience to form evenly spread film. Thermo BioMate 3S UV–visible spectrophotometer was used for the UV–Vis absorption measurements, spectra were typically measured in the range of 200–700 nm. Photoluminescence spectra were carried out using SHIMADZU RF-5301 PC instrument. Cells fluorescence imaging were collected by laser scanning confocal microscope Carl Zeiss LSM710 (Zeiss, Germany). Nude mice in vivo imaging were carried on vivo multispectral imaging system (Maestro EX).
Preparation of glutathione stabilized Au nanoclusters and Au/Ce nanoclusters
Precisly prepared 2.4 mM glutathione aqueous solution, measured out 8.5 μL pre-prepared 1 M HAucl4 aqueous solution and added into 5 mL 2.4 mM GSH solution, and shaked vigorously until the solution becomes colorless. The final concentration of HAuCl4 was 1.7 mM. Then the mixture was placed in a water bath with 90°C for two hours to obtain low fluorescence Au seed crystal and added 5 μL 1 M Ce(NO3)3 aqueous solution immediately. The mixture was blending and heating in water bath with 90°C for another one hour. A illustration need to be added that the synthesis of pure Au nanoclusters were performed in same condition without adding cerium ion. The products were separed and purified by centrifugation at 10000 rpm for 10 min with 1:4 ethanol. The precipitation were redispersed in deionised water or phosphate buffer solution (PBS, pH 7.2) for further application.
Cell growth inhibition study by MTT assay
Briefly, 100 μL medium with 2 × 103 Cells/well were plated in 96-well plates, after about eight hours incubation, cells were treated with 100 μL various concentrations of Au/Ce NCs medium solution. Each concentration set up five repetition. After treatment for 24 hours, 20 μL MTT solution (5 mg/ml) was added to each well. And cells were incubated for another four hours, then the supernatant was removed and 150 μL DMSO was added per well. Samples were then shaked well for 10 min and the optical density (OD) was read at a wavelength of 490 nm by microplate reader (MK3, ThermoFisher). All experiments were performed in triplicate.
Construction of the xenografted tumor mouse model
BALB/c female athymic nude mice, age-matched (four weeks of age) and weight-matched (18–22 g), were purchased from Peking University Health Science Center. All experiments involving mice were approved by the National Institute of Biological Science and Animal Care Research Advisory Committee of Southeast University, and experiments were conducted following the guidelines of the Animal Research Ethics Board of Southeast University. The mice were randomly assigned to groups for experimental purposes. They were maintained in clean facilities with a 12-hour light/dark cycle and received water and food through a semi-barrier system. Subcutaneous tumor models were generated by the subcutaneous inoculation (0.10 mL volume containing 5 × 107 cells/mL media) of HeLa cells in the right side of their armpit or nearby sites using a 1-mL syringe with a 25 G needle. Tumor growth was monitored until a palpable size for next applications.
In vitro and in vivo bioimaging study
For cellular imaging, HeLa cells and L02 were treated with a certain concentrations of Au/Ce NCs solutions and incubated at 37°C for 24 h. The cells were washed three times with PBS before fluorescence imaging. A 488-nm excitation laser beam (Andor Revolution XD) was focused using a 63× IR coated objective (Nikon). Similarly, HepG2 cells were treated with 150 μmol/L Au/Ce NCs solutions, the cells were washed three times with PBS before fluorescence imaging. A 488-nm excitation laser beam (Andor Revolution XD) was focused using a 20× IR coated objective (Nikon).
For in vivo bio-imaging of Au/Ce NCs in the tumor location complex solution was administered into the solid tumor mouse model through local injection or intravenous injection through the tail. The mice were fully anesthetized by gaseous 5% isoflurane anesthesia. The in vivo bio-images were acquired on Cri Maestro in vivo imaging system. After incubation for 24 h, fluorescent Au/Ce nanoclusters were observed inside the tumors by in vivo fluorescence imaging using a 455 nm excitation wavelength. In comparison, the negative control groups, which received an equivalent volume of Phosphate Buffered Saline (PBS), did not exhibit any apparent fluorescence. The ROI (regions of interest) analysis was measured under the assistance of CRi Maestro Image software. The studies were approved by the National Institute of Biological Science and Animal Care Research Advisory Committee of Southeast University, while experiments conducted the guidelines of the Animal Research Ethics Board of Southeast University.
This work is supported by National Natural Science Foundation of China (81325011), National High Technology Research and Development Program of China (2012AA022703) and the National Basic Research Program (2010CB732404) as well as Suzhou Science & Technology Major Project (ZXY2012028).
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