Chinese Chemical Letters  2015, Vol.26 Issue (11): 1400-1402   PDF    
Novel in situ biosynthesized fluorescent zinc nanoclusters for specific cellular bio-imaging
Mei-Na Su, Jing Ye, Qi-Wei Li, Shui-Hong Li, Wei Ge, Xiao Chen, Hui Jiang, Xue-Mei Wang     
State Key Laboratory of Bioelectronics (Chien-Shiung Wu Lab), School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China
Abstract: It is well known that zinc ions play an indispensable role in the structure and function of a large number of biological process and relevant bio-macromolecules. When some cancers occurred, the relevant concentration of zinc ions considerably decreased. Since cancer cells have a completely different redox homeostasis from normal cells, in this contribution, we have explored the possibility of bio-imaging or labeling of cancer cells through the in situ biosynthesized zinc nanoclusters by cancerous cells. The results demonstrate that we can readily realize the in vivo fluorescent bio-imaging of cancer cells through the in situ biosynthesis of the biocompatible zinc nanoclusters from cancerous cells (i.e., Hela cervical carcinoma cell line and others) when target cells cultured with micromolar zinc gluconate solutions.
Key words: Flourescent bio-imaging of cancer cells     Biosynthesis     Zinc nanoclusters     Zinc ions    
1. Introduction

Most of cancers could not be diagnosed in the early stage so that many patients lost the best time of effective treatment. Thus,it is important to develop new diagnostic methods to enable the accurate diagnosis of cancers in the early stage [1, 2, 3, 4, 5]. In recent years,different kinds of strategies have been explored for cancer early diagnosis [6, 7, 8]. Among them,medical imaging has become an indispensable diagnosis tool and fluorescence imaging of tumor locations may accurately localize the diseased sites for the direct bio-imaging of cancer cells and relevant tissues [9, 10]. With the development of nanotechnology,a wide range of nanomaterials, including semiconductor quantum dots like ZnO NPs [11, 12],ZnS QDs [13] and others,have been explored in cancer bio-imaging. Our recent studies demonstrate that bio-synthesized fluorescent gold or silver nanoclusters (NCs) could be readily utilized as a great potential probe for highly sensitive optical imaging of cancers [14, 15].

It is well known that zinc ions play an indispensable role in the structure and function of a large number of biological process and relevant bio-macromolecules [16, 17]. When some cancers occurred,the relevant concentration of zinc ions considerably decreased,accompanied with relatively insufficient glucose and low oxygen concentration levels,which will give rise to a more reducing environment compared to normal cells/tissues. Based on these considerations,in this contribution we have explored the possibility of bio-imaging or labeling of cancer cells through the in situ biosynthesized zinc nanoclusters by cancerous cells. The observations demonstrate that since cancer cells have a completely different redox homeostasis from normal cells,we can readily realize the in vivo fluorescent bio-imaging or labeling of cancer cells through the in situ biosynthesis of the biocompatible zinc nanoclusters from cancerous cells (i.e.,Hela cervical carcinoma cell line and others) when cultured with micromolar zinc gluconate solutions.

2. Experimental

Hela cells (cervical carcinoma) were purchased from Shanghai Institute of Cells,Chinese Academy of Sciences. L02 cells (human embryo liver cell strand) were supplied by Third Military Medical University (Chongqing,China).

Hela and L02 cells were cultivated in DMEM (high glucose, Hyclone) medium including with 10% fetal calf serum (Sigma, USA),100 U/mL penicillin (Sigma,USA),and 100 mg/mL streptomycin (Sigma,USA) at 37 ℃ with 5% CO2 in a 95% humidified atmosphere.

Hela and L02 cells in log phase were trypsinized and then cultured in 6-well plates (1 × 105 cells per well). Each well was added into 0.08 mmol/L Zn(C6H11O7)2 solution. Laser confocal fluorescence microscopy was utilized to complete confocal luminescence microscopy imaging of these cells.

Zinc gluconate (Zn(C6H11O7)2) was obtained from Sigma,and stored in the dark. Aqueous solution of zinc gluconate (Zn(C6H11O7)2) was freshly prepared and used for the biosynthesis of zinc nanoclusters (Zn NCs). The in situ biosynthesized zinc nanoclusters were harvested from the cancer cell lines like Hela cells incubated with DMEM contained 0.08 mmol/L Zn(C6H11O7)2 for 24 h at 37 ℃ with 5% CO2 by repetitive freeze-thaw method.

A JEM-2100 transmission electron microscope was used to obtain the TEM images. The confocal luminescence imaging in cells was investigated by laser confocal fluorescence microscopy (Leica TCS SP2). The excitation wavelength of the fluorescence was 488 nm.

3. Results and discussion

Initially,we have explored the possibility of bio-imaging or labeling of cancer cells through the in situ biosynthesized zinc nanoclusters (NCs) by cancerous cells. Hela cancer cells and other cell lines were selected as models to evaluate the pertinence of the biosynthesis of zinc NCs in situ by cancer cells. To compare,normal cell lines (i.e.,L02,liver cells) were set as control. As shown in Fig. 1 and Fig. 2,the relevant cells were incubated for 24 h with micromolar zinc gluconate (Zn(C6H11O7)2) solutions,then the intracellular presence of the ensuring biosynthesized zinc NCs were characterized by fluorescence microscopy,transmission electron microscope (TEM) together with energy dispersive spectroscopy (EDS). A typical TEM image of the biosynthesized Zn NCs was showed in Fig. 1(A),demonstrating that they have high monodispersity and their uniform sizes are 2-3 nm. At the same time,EDS was used to analyze the elemental in the zinc NCs, illustrating that no other elemental impurity was present in the biosynthesized zinc NCs. Meanwhile,it is observed that under excitation at 415 nm,the biosynthesized Zn NCs exhibited emission bands at 640 nm.

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Fig.1. (A) Transmission electron microscopy and EDS spectroscopy (B) the zinc nanoclusters biosynthesized in situ by Hela cancer cells incubated for 24 h with Zn(C6H11O7)2.

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Fig.2. Laser confocal fluorescence micrographs of L02 cells (A) and Hela cells (B) which were incubated withDMEM containing identical 0.08 mmol/L Zn(C6H11O7)2 for 24 h at 37 ℃.

Fig. 2 illustrates the typical intracellular images of confocal fluorescence microscopy which were applied to evaluate the bioimaging of cancer cells cultured with DMEM (high glucose) contained 0.08 mmol/L Zn(C6H11O7)2. As to be expected,the intracellular fluorescence was obviously detectable when cancer cells like Hela cell lines cultured in DMEM medium which contained 0.08 mmol/L Zn(C6H11O7)2 (Fig. 2B). For control experiments, no observable intracellular fluorescence signal could be observed when the equivalent concentration of Zn(C6H11O7)2 were added to the non-cancerous cells culture (i.e.,L02 cells) (Fig. 2A), indicating that this biosynthesis of Zn nanoclusters from zinc gluconate solutions could not occur in non-cancerous cells and seems only specific to the cancerous cell model,thus allowing kinetically-controlled selective imaging of cancer cells by fluorescence microscopy.

4. Conclusion

This study demonstrates a new strategy of the biosynthesis of Zn nanoclusters from zinc gluconate solutions for targeting bioimage of cancer cells. The biosynthesized zinc NCs can only accumulate in cancer cells and not appear in non-cancerous cells. The rationale behind it can be attributed to the fact that when some cancers occurred,the relevant concentration of zinc ions considerably decreased,accompanied with relatively insufficient glucose and low oxygen concentration levels,which will lead to a completely different redox environment compared to normal cells. Thus,by taking the advantage of the relevant peculiarities of cancer cells,we can readily realize in situ bio-synthesized fluorescent Zn nanoclusters for highly sensitive optical imaging of cancer cells. This raises the possibility of specific fluorescent self-bio-marking of target cells and tumor locations for the promising fluorescence imaging-guided therapy of tumors in future clinical applications.

Acknowledgments

This work is supported by the National Natural Science Foundation of China (No. 81325011),National High Technology Research & Development Program of China (No. 2015AA020502, 2012AA022703),and the Major Science & Technology Project of Suzhou (No. ZXY2012028).

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