Highly Selective Red-Emitting Fluorescent Probe for Imaging Cancer

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High-Selective Red-emitting Fluorescent Probe for Imaging Cancer Cells in Situ by Targeting Pim-1 Kinase Shigang Guo, Jiangli Fan, Benhua Wang, Ming Xiao, Yueqing Li, Jianjun Du, and Xiaojun Peng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14553 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 8, 2017

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ACS Applied Materials & Interfaces

High-selective Red-emitting Fluorescent Probe for Imaging Cancer Cells in Situ by Targeting Pim-1 Kinase Shigang Guoa, Jiangli Fan*a, Benhua Wanga, Ming Xiaoa, Yueqing Li b, Jianjun Du a, Xiaojun Penga a

State Key Laboratory of Fine Chemicals, bSchool of Pharmaceutical Science and Technology,

Dalian University of Technology, Dalian, 116024, China. *email: [email protected]

ABSTRACT: Based on the fact that the enzyme-targeting probes were high sensitive and selective, a novel red-emitting probe (NB-BF) for Pim-1 kinase including three parts: fluorophore (NB), linker and inhibitor (BF), was designed for cancer optical imaging. In its free state, NB-BF was folded and the fluorescence was quenched by PET between fluorophore and inhibitor both in PBS buffer and normal cells. Significantly, it emitted strong red fluorescence in Pim-1 over-expressed cancer cells. The specificity of NB-BF for Pim-1 kinase was directly demonstrated by gene silencing analysis. Furthermore, it is the first time to know where Pim-1 kinase mainly distributes at mitochondria with Pearson’s correlation factor (Rr) of 0.965 and to provide a fluorescent tool to verify the function of the Pim-1 kinase. More importantly, NB-BF was applied in tissue imaging and preferentially labeled the tumors in vivo.

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KEYWORDS: Pim-1 kinase, Biomarker targeting, Cancer cell imaging, Mitochondria localization, in vivo imaging

1. INTRODUCTION

Cancer becomes one of the most mortally diseases during recent years in spite of rapid advances made in diagnostic and the treatment procedures.1,2 It is critical and essential to single out new targets and establish novel techniques for accurate cancer detection. Most cancer-associated proteases are intracellular enzymes and some of them turn out to be overexpressed in cancer cells.3-5 The Pim serine protein kinases, potential causative enzymes, are reliable diagnostic markers for cancers because they play crucial roles in the growth and progression of multiple cancer types.6-8 Among Pim protein kinases, Pim-1 kinase is richly expressed and found to be secreted by diverse kinds of cancers, including head and neck cancer,9 prostate cancer,10 breast cancer,11 and gastric carcinoma12. The clinical data have confirmed that elimination of Pim-1 gene has no evident effect on normal cells and yet is pernicious to cancer cells, thus making Pim1 a potentially useful target for rational cancer therapy, and in some cases, it can act as a prognostic indicator in some clinical research.13-15 Owing to its close link with cancer, the detection of Pim-1 has attracted considerable attention and effort in recent years.16 Different methods, such as western blot, immunohistochemical analysis, magnetic resonance imaging (MRI), positron emission tomography (PET) and semi-quantitation RT-PCR based assay, have been used to research the contribution of Pim-1 to tumor development and progression.9,17-22 Although these methods could satisfy the detection of Pim-1 in solution and biological fluids, they are not suitable in living bio-systems because they usually need a lot of pretreatment processes which would result in damage to living samples. Besides, they carry a lot


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of drawbacks, such as high instrument cost, limited spatial resolution, and radiological hazards.23, 24

Recently, fluorescence imaging technology has play a dominant role in the localization and

further dynamic monitoring of diseases-related biomarkers as a consequence of the low-cost with high sensitivity and spatial resolution, and non-damage real-time imaging capabilities.25 Considering some enzymes are overexpressed in cancer cells, a few enzyme-targeted probes have been created to distinguish carcinoma cells from normal cells.26,

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Nonetheless, the

absorption and emission peaks of these sensors are located in the visible range, which limits their applications to a certain extent. It is known to us that long wavelength light (> 600 nm) has considerable advantages such as minimal interference caused by background autofluorescence, minimum photo-damage, and adequate penetration of the emissive light through biological samples. Along these lines, we need to pay much attention to red-emitting or near infrared (NIR) fluorescent probes.28- 32

Figure 1. Recognition mechanism of NB-BF.

Bearing these facts in mind, we design a fluorescence turn-on Pim-1-specific probe, NB-BF. In this probe, a powerful inhibitor of Pim-1 kinase, 5-bromobenzofuran-2-carboxylic acid, was bond to a red-emitting fluorophore Nile blue by a hexanediamine linker (Figure 1). NB-BF can not only bind Pim-1 kinase in suit among cancer cells but also distinguish cancer cells from


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normal ones. Particularly, it could apply in tissue imaging and preferentially label the tumors in vivo.

2. RESULTD AND DISCUSSION

2.1 Design and Preparation of NB-BF

Photoinduced electron transfer (PET)33 represents the main principle behind the design of small molecule “off-on” fluorescent probes. To realize “off-on” fluorescence for enzymes, a unique design platform was developed, in which PET was formed through intramolecular space folding.34-37 This design platform overcame the difficulty of designing PET enzyme-targeting probes and was more suitable for detecting biomarker in suit. Thus in this work, the fluorophore was chosen as Nile blue due to its long excitation and emission wavelength, high fluorescent quantum yields, and good photo-stability. 5-bromobenzofuran-2-carboxylic acid (BF) 38, a good inhibitor for Pim-1 kinase with IC50 = 8.5 µM, was linked to Nile blue by a linear hexanediamine which made NB-BF easy to be folded in its free state. Once BF inserted fully into the hydrophobic pocket of Pim-1 kinase, NB-BF was forced to be unfolded. The target molecule was synthesized on the basis of the route shown in Scheme 1. NB-BF was obtained in 61% yield according to the amide condensation of 5-bromobenzofuran-2-carboxylic acid with Nile blue under EDCl, DMAP and HOBT for 24 h. The structure of NB-BF was well characterized by 1H NMR, 13C NMR and TOF-MS (Figure S10-S12).

2.2 Mechanism of the Fluorescence “off-on”


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For understanding the fluorescence “off-on” mechanism, the molecular structure of NB-BF was optimized. Taken advantage of Gaussian 09 (DFT at the B3LYP/6-31G level), the frontier molecular orbital energies of NB-BF were also calculated in water (Figure 2). The distance between NB and BF was determined as about 1.0 nm indicating that NB-BF remained a folded state. And the oscillator strength of HOMO to LUMO transition was only 0.059, which means the electron transition was prohibited. i.e. in the folded state, the fluorescence of NB-BF was quenched by PET between the dye and the inhibitor part (BF).

Figure 2. Frontier molecular orbital of NB-BF calculated with Gaussian 09.

Furthermore, in an effort to imitate the probable interaction, NB-BF was docked with Pim-1, using CDockermodule in Discovery Studio 2.5.39 The X-ray crystal structure of Pim-1 compounded with 5-bromo-1-benzofuran-2-carboxylic acid (PDB code 3R00) was collected from the PDB database (http://www.rcsb.org/pdb).38,40 The center of binding site was defined based on 5-bromo-1-benzofuran-2-carboxylic acid and adjusted according to the narrow groove shape. The 3D coordinate of the sphere around the center of the binding site was -14.76, -33.14, 1.28 (Figure 3a). The radius of the sphere was 11.0 Å. From the result of CDOCKER, the BF


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part and 5-bromo-1-benzofuran-2-carboxylic acid were in the similar position (Figure 3b). The whole stretched probe could be docked well into the narrow groove (Figure 3c). Interactions between the hydrophobic regions of a binding site are often observed to provide the driving force for binding.41 In the crystal structure (3R00), the bromo group is located in the hydrophobic pocket surrounded by A65, R122, L44 and L174, and the 2-carboxylic acid group forms the saltbridge interaction with K67.28. Therefore, although the inhibitor was modified, 5-bromobenzofuran group of the probe is still in the hydrophobic pocket in the docking result. The long chain lies along the cranny and fits well (Figure 3c). These results showed that NB-BF would be an unfolded state when the targeting part was embedded in the groove of Pim-1 kinase. Hence, the PET process was inhibited by the long hexanediamine linker between NB fluorophore and BF inhibitor, and thus the fluorescence was restored.

Figure 3. Docking result of NB-BF in the binding site of Pim-1. (a) The sphere of binding site in 3R00. (b) Docked poses of NB-BF in 3R00. (c) A stretched pose of NB-BF lies in the groove of Pim-1. The carbon atoms of 5-bromo-1-benzofuran-2-carboxylic acid are green shown in stick mode. The carbon atoms of the NB-BF are yellow shown in stick mode.

2.3 Spectroscopic Properties


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The detailed spectroscopic and photophysical properties of NB-BF in different solvents were listed in Figure S1 and Table S1. It is important that NB-BF was almost non-fluorescent with the fluorescence quantum yield of 0.005 in PBS aqueous solution. And it exhibited such weak fluorescence in a wide pH range from 1-13 (Figure S2), which indicated that NB-BF could be applied in a wide pH range. These properties made NB-BF have good signal-to-noise ratio in the bio-imaging and avoid the interference of intracellular pH change. The interference experiment of NB-BF was also performed in PBS buffer solution (pH 7.4, 60 mM) with common species, such as amino acids, cations and anions, which are often found in environmental and biological settings (Figure S3). The result showed that no observable fluorescence response occurred in the presence of these common species, indicating well anti-interference capability of the probe.

2.4 Water solubility, Photostability and Cytotoxicity

As shown in Figure S4, there is a good linear relationship between the absorbance and the 0-8.0 µM of NB-BF in PBS buffer solution (pH 7.4, 60 mM), which means that aggregation did not happen under our experiment conditions. From the photostability test (Figure S5), the fluorescence intensity of NB-BF remained about 90% in PBS solutions irradiated for 4 h with a 500 W iodine-tungsten lamp. Evaluated via the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2-H-tetrazolium bromide) test, low cytotoxicity of NB-BF was detected (Figure S6). These attractive properties made the probe’s application in living specimens possible.

2.5 Fluorescence imaging in living cells

The concentrations in different cancer cells and normal cells were verified by enzyme-linked immunosorbent assay (ELISA). There were obvious expression differences between cancer cells


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(MCF-7 cells 5.92 ng/ml and HepG2 cells 2.85 ng/ml) and non-cancer cells (COS-7 cells 1.43 ng/ml and RAW264.7 cells 1.086 ng/ml), which indicated that Pim-1 kinase is an applicable biomarker for cancer detection. Upon treatment with 2.5 µM NB-BF in MCF-7 cells, the fluorescence images were recorded at intervals by a confocal fluorescence microscope. As shown in Figure S7, the addition of NB-BF caused time-dependent fluorescence enhancement and the fluorescence intensity increased to the maximum at 30 min, and then kept stable after prolonged incubation time (150 min). Next, two cancer cell lines (MCF-7 and HepG2 cell) and two non-cancer cell lines (COS-7 and RAW246.7 cell) were incubated with 2.5 µM NB-BF for 30 min and imaged immediately. As shown in Figure 4, upon excitation at 635 nm, the two cancer cell lines exhibited strong fluorescence, whereas normal ones showed negligible fluorescence. Therefore, NB-BF could serve as a promising visual tool to distinguish cancer cells from normal cells.


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Figure 4. (a) Fluorescence images of different cells staining with 2.5 µM NB-BF. λex = 635 nm, detection window = 640-700 nm. (b) Quantitative image analysis of the fluorescence intensity of different cells. Each value is the average of eight cells in each sample image.

Gene silencing analysis was performed to verify the specificity of NB-BF for Pim-1 kinase. The plasmid vector (purchased from Shanghai Jikai Gene Company) contains shRNA and fluorescent protein genes. As shown in Figure 5a, after the plasmid vector was transfected to MCF-7 cells, along with the hairpin of shRNA digesting into siRNA, the fluorescent protein was expressed with strong blue fluorescence, while siRNA inhibited the expression of Pim-1 kinase and red fluorescence of NB-BF was depressed. Furthermore, how much of the plasmid vector into cells by cell phagocytosis will influence the gene silencing effect. Therefore, in Figure 5b, with the increment of plasmid into cell 1, cell 2 and cell 3 respectively, the blue fluorescence from fluorescent protein increased while the red fluorescence from NB-BF decreased gradually. All these results clearly indicated that NB-BF specifically targeted to Pim-1 kinase and the fluorescence of probe associated with the abundance of Pim-1 kinase. Besides, HepG2 cells were pre-incubated with 0, 5.0, and 10.0 µM 5-bromobenzofuran-2-carboxylic acid for 5 h respectively, and then treated with NB-BF for 30 min. The fluorescence intensity decreased gradually with increasing amount of inhibitor (Figure 6), which illustrated the specificity of NBBF for Pim-1 kinase complementally.


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Figure 5. Fluorescence imaging of siRNA interference assay. (a) Fluorescence images of MCF-7 cells pre-treated with shRNA and then 2.5 µM NB-BF. Blue emission from siRNA, λex = 488 nm, detection window = 500-540 nm, red emission from NB-BF, λex = 635 nm, detection window = 640-700 nm; (b) Quantitative image analysis of fluorescence intensity in cell 1, cell 2 and cell 3.


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Figure 6. Special recognition of Pim-1 by NB-BF. (a) Competitive fluorescence images of HepG2 cells pre-treated with 0, 5.0, or 10.0 µM 5-bromobenzofuran-2-carboxylic acid prior to NB-BF (2.5 µM) treatment. λex = 635 nm, detection window = 640-700 nm; (b) Quantitative image analysis of the fluorescence intensity in cells. Each value is the average of eight cells in each sample image.

2.6 Specific localization of cancer cells

To determine the subcellular distribution of NB-BF, nuclei specific staining dye Hoechst 33258 (1.0 µM) was used to co-stain MCF-7 cells with NB-BF (2.5µM). The images (Figure 7) showed that only minimal co-localization of the red emissions of NB-BF with blue emissions from the Hoechst33258, indicating that NB-BF distributed mainly in the cytoplasm region which is identical with the previous reports that Pim-1 kinase mainly accumulates in the cytoplasm.42


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Nevertheless, Pim-1 kinase’s specific subcellular distribution is rarely reported. It has been reported that Pim-1 is a crucial regulator which participates in hypoxia-induced chemoresistance, which means that hypoxia can increase Pim-1 expression.43 Given that mitochondria is regarded as oxygen concentration sensor, and it plays pivotal role in the adaptation of organism to hypoxia. Therefore, it is conceivable that Pim-1 would accumulate preferentially in the mitochondria of cancer cells. In order to confirm whether NB-BF could be used to image mitochondria in the cytoplasm region, a commercial probe for mitochondria, Mito-Tracker Green (1.0 µM), was used to co-stain MCF-7 cells with NB-BF (2.5 µM). The co-localization coefficient was assessed by Pearson’s correlation factor (Rr)34. The green fluorescence images (Figure 8b) stained by Mito-Tracker Green matched well with the red fluorescence images stained by NB-BF (Figure 8c), Rr = 0.965 (Figure 8e). We selected a region of interest (ROI) of the overlay image, and conducted curve fitting of the two channels, as shown in Figure 8f, the distribution of fluorescence intensity in the ROI was basically similar. The similar results (Figure S8) were obtained in HepG2 cells with Rr = 0.957. Moreover, we also co-stained various subcellular organelles of MCF-7 cells, including lysosome (Lyso-tracker Green), endoplasmic reticulum (ER-tracker Green), and Golgi apparatus (NBD-C6-ceramide) with Rr = 0.752, 0.744, 0.795, respectively (Figure S9). These observations indicated that Pim-1 kinase mainly distributed at mitochondria. Significantly it is the first time to know where Pim-1 kinase distributes in cytoplasm and to provide a fluorescent tool to verify the function of the Pim-1 kinase.


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Figure 7. Costain images of NB-BF (2.5 µM, λex =635 nm, detection window = 640-700 nm), and Hoechst 33258 (1.0 µM, λex = 405 nm, detection window = 420-450 nm) in MCF-7 cells. (a) bright field image; (b) Blue emission from Hoechst 33258; (c) Red emission from NB-BF; (d) Overlay of the blue and red channels.

Figure 8. Fluorescence images of NB-BF (2.5 µM) in MCF-7cells co-stained with Mito-Tracker Green (1.0 µM). (a) bright field image; (b) green emission from Mito-Tracker Green, λex = 488


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nm, detection window = 500-540 nm; (c) red emission from NB-BF, λex = 635 nm, detection window = 640-700 nm; (d) overlay of the green and red channels; (e) colocalization analysis; (f) Intensity profile of region cross co-stain image.

2.7 Fluorescence images in tissue slices

Figure 9. Fluorescent images of NB-BF (8 µM) stained in live tissues for 5 min at 37°C. (a, b) are normal tissues. (c, d) are breast cancer tissues. (a, c) Fluorescent images, (b, d) Bright-field images. λex = 635 nm, detection window = 640-755 nm. (e) Quantitative image analysis of the average fluorescence intensity in two tissues.

Then NB-BF (8 µM) was incubated with cancerous and normal tissue slices respectively to further investigate the applicability of NB-BF for tissue imaging. As shown in Figure 9, the cancer tissue showed strong fluorescence signals compared to those in normal tissue slices (Figure 9), suggesting that NB-BF could figure out the cancerous tissues from normal ones by fluorescence imaging.


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2.8 Fluorescence imaging in vivo Finally, the in vivo tumor fluorescence tests were performed using a small animal living imaging system ( λex = 630 nm, λem = 700 nm). After nude mice carry MDA-MB-231 xenografts were treated with NB-BF 200 µM/100 µL PBS via subcutaneous injection for 30 min, significantly, strong fluorescence signals were detected in the tumor site (Figure 10) which illustrated that NBBF was suitable for tumor labelling in vivo.

Figure 10. The white light image and fluorescence image of nude mice with the MDA-MB-231 tumor. Mice were treated with NB-BF 200 µM/100 µL PBS via subcutaneous injection and incubated for 30 min.

3. CONCLUSION

To summary, the first Pim-1-specific red-emitting fluorescent probe (NB-BF) was reported for visualizing cancer cells. It showed an off-on fluorescence change due to the inhibition of PET process when NB-BF bond to Pim-1. NB-BF has good water solubility, high photostability and


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relatively low cytotoxicity. The specificity of NB-BF for Pim-1 kinase was directly demonstrated by gene silencing analysis. Consequently, NB-BF was used to analyze distribution of Pim-1 in the cytoplasm and firstly speculate the Pim-1 kinase mainly in the mitochondria. More importantly, NB-BF could distinguish tumor tissues from normal tissues and fluorescently image tumor in vivo. Taken these in concert, we anticipate that NB-BF might be a potential tool for the early diagnosis of cancers and fluorescence surgery.

4. MATERIALS AND METHODS

4.1 Synthesis of NB-BF

Scheme 1. Synthetic procedures of NB-BF.

(1) Preparation of NB: NB was synthesized from 5-isopropyl-2-nitrosophenol and N(naphthalen-1-yl) hexane-1,6-diamine by the procedure published in the literature33. 1H NMR (400 MHz, CD3OD), δ: 8.94 (d, J = 8 Hz,1H), 8.42 (d, J= 8 Hz, 1H), 7.96 (m, 1H), 7.88 (m, 2H), 7.29 (m, 1H), 7.04 s, 1H), 6.92 (d, J = 2.8 Hz, 1H), 3.79 (t, J =8 Hz, 2H), 3.33 (s, 6H), 2.96 (t, J = 8 Hz, 2H), 1.93-1.56 (m, 8H). 13C NMR (100 MHz, CD3OD), δ:25.8, 26.2, 27.1, 28.1, 39.3, 39.8, 44.3, 95.6, 93.2, 115.0, 122.8, 123.1, 124.0, 129.6, 129.9, 130.6,131.6, 132.1, 133.3, 147.3, 151.3, 155.5, 157.7 ppm; HRMS: m/z calcd for C24H29N4O+ [M]+: 389.2336, found: 389.2323


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(2) Preparation of NB-BF: Nile blue (120 mg, 0.308 mmol), 5-bromobenzofuran-2-carboxylic acid (74.24 mg, 0.308 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 70 mg, 0.370 mmol), HOBt·H2O (70 mg, 0.462 mmol) and 4-dimethylaminopyridine (DMAP, 45 mg, 0.370 mmol) were added to 10 ml dehydration DMF solution. The mixed solution was stirred at room temperature under nitrogen for 24 h and then concentrated. The residue was purified by flash chromatography on silica gel withCH2Cl2/CH3OH = 20:1, affording the desired violet solid NB-BF (115 mg, 61%). 1H NMR (400 MHz, DMSO-d6), δ: 8.80 (d, J = 4 Hz, 1H), 8.60 (d, J = 4 Hz, 1H), 7.98 (t, J = 8 Hz, 2H), 7.87 (t, J = 4 Hz, 1H), 7.71 (d, J = 4 Hz, 1H), 7.57 (m, 2H), 7.45 (s, 1H), 7.40 (t, J = 8 Hz, 1H), 7.25 (d, J = 8 Hz, 1H), 7.06 (s, 1H), 6.86 (s, 1H), 3.74 (t, J = 4 Hz, 2H), 3.27 (s, 6H), 1.78 (t, J = 8 Hz, 2H), 1.58-1.23(m, 8H); 13C NMR (100 MHz, DMSO-d6), δ: 157.6, 154.9, 152.9, 151.4, 150.4, 147.2, 142.8, 133.9, 131.9, 130.6, 129.6, 129.3, 129.2, 127.8, 126.9, 125.0,124.3, 123.9, 119.0, 115.8, 114.8, 113.7, 109.7, 108.4, 95.7, 93.7, 44.3, 40.5, 38.6, 28.9, 28.2, 26.0 ppm; TOF MS: m/z calcd for C33H32BrN4O3+[M]+: 613.1652, found: 613.1623.

4.2 Determination of fluorescence quantum yields

The relative fluorescence quantum yields of NB-BF in different kinds of solution were performed according to the following equation.

ϕu =

(ϕ s )( FAu )( As )(λ exs )(η u2 ) ( FAs )( Au )(λ exu )(η s2 )

Here, φ = fluorescence quantum yield; FA = integrated area of the corrected emission spectra; A = absorbance at the excitation wavelength; λex = excitation wavelength; η = refractive index of


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the solution, and the subscripts s and u mean the standard and the unknown, respectively. Rhodamine B, the fluorescence quantum yield is 0.49 in ethanol was chosen as standard.

4.3 Photostability experiments

NB-BF, Niblue and Mito-Tracker Green were dissolved in PBS buffer (60 mM, pH 7.4) all at concentrations of 5.0 µM. The photostability experiment was performed by placing these sample solutions in square quartz cells and then irradiating the samples at a distance of 300 mm away for 4 h under a 500 W iodine-tungsten lamp. To keep the light shorter than 400 nm and eliminate heat, a saturated NaNO2 aqueous solution (50 g/L) was set up between the lamp and samples. The photostabilities were expressed in terms of remaining fluorescence intensity (%), which were calculated from the fluorescence changes at the maximum absorption wavelength before and after irradiation by iodine-tungsten lamp. All samples were tightly sealed. 4.4 Water solubility and pH stability A UV-Vis spectrophotometer of aqueous solution of NB-BF with different concentrations (012.5 µM) were measured by HP 8453-UV3100 spectrophotometer (Agilent, USA). The concentration gradient Tris-HCl buffer was titrated using a precision pH meter. In the presence of NB-BF (5 µM), the fluorescence intensities at different pH values were obtained by a FP-6500 fluorescence spectrophotometer (Jasco, Japan). 4.5 Enzyme-Linked Immunosorbent Assay (ELISA) ELISA experiments were implemented commercially by Dalian Medical University. The Pim-1 kit (human, double-antibody method, 96t) was purchased from Abcam Co. (USA). Cells were homogenized and stored at 2-8 °C.


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4.6 Cell incubation and staining with NB-BF

The mammalian cells MCF-7 (human breast cancer cells), HeLa (Human cervical cancer cells), HepG2 (human hepatoma cells), COS-7 (African green monkey kidney cells) and Raw 264.7 (mononuclear macrophage cells) were obtained from Institute of Basic Medical Sciences (IBMS) of the Chinese Academy of Medical Sciences. All the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) which was supplemented with 10% fetal bovine serum and 1% gentamicin sulphate. The cells were cultivated in 24-well flat-bottomed plates and then incubated for 24 h at 37 ºC under 5% CO2. 2.5 µM NB-BF were added into the plates to incubate for another 30 min and then washed with phosphate-buffered saline solution (PBS) three times. The fluorescence images were achieved by an OLYMPUSFV-1000 inverted fluorescence microscope with a 60×objective lens. The stain images were obtained using excitation and emission wavelengths at 635 nm and 640-700 nm, respectively.

4.7 Fluorescence counterstaining of live cells

Mito-Tracker Green (1.0 µM), Lyso-Tracker Green (1.0 µM), ER-Tracker Green (1.0 µM) and NBD-C6-ceramide (2.5 µM) were used to co-stain the cells. Cells were incubated for 30 min at 37 ºC under 5% CO2 and then washed with PBS three times. Fluorescence images were then carried out with OLYMPUSFV-1000 inverted fluorescence microscope, using a 60×objective lens. NB-BF (red channel) was excited at 635 nm, and the emission spectra were collected at 640-700 nm. The commercially available probes of subcellular organelles (green channel) were excited at 488 nm, and the emission spectra were collected at 500-540 nm.

4.8 Gene silencing analysis


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The plasmid vector of shRNA correctly measured sequence was purchased from Shanghai JiKai Gene Company. The mammalian cells MCF-7 were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% gentamicin sulphate. Then the MCF-7 cells were cultivated in confocal special dish and treated with the plasmid vector for 72 h. The completeness of transfection was testified via fluorescence microscopy. After transfection, cells were incubated with 2.5 µM NB-BF for another 30 min and then washed with PBSthree times. Fluorescence images were collected using fluorescence microscope with a 60×objective lens. The stain images were obtained using excitation and emission wavelengths at 635 nm and 640-700 nm (red channel), 488 nm and 500-540 nm (blue channel), respectively.

4.9 Cytotoxicity experiments

Cell viability was evaluated by MTT assay. MCF-7 cells were cultivated in 96-well microplates (Nunc, Denmark) at a density of 1×105 cells/mL in 100 µL medium supplemented with 10% FBS. The plates were washed with 100 µL/well PBS after 24 h attachment. And then 2.5 µM NB-BF was added to the medium and incubated for 24 h. At the meantime, cells in culture medium without treating NB-BF were used as control. For each control and test concentration, six replicate wells were performed. After that, 10 µL PBS which contains MTT (5 mg/mL) was added to each well. After another 4 h incubation at 37 ºC in a 5% CO2 humidified incubator, medium was carefully removedand the purple crystals were lysed in 200 µL DMSO. Finally, the optical density was measured on a microplate reader (Thermo Fisher Scientific) at 570 nm with deduction of the absorbance of the blank well at 630 nm. Cell viability was performed using the following equation: Cells viability (%) = (ODdye–ODKdye) / (ODcontrol–ODKcontrol) × 100%


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Here dye means the sample containing NB-BF, control is control group, and the blank group is denoted as K.

4.10 Preparation of tissue slices and staining with NB-BF

The breast cancer tissues were from clinical human breast cancer patients (Department of Pathology, First Affiliated Hospital of Dalian Medical University). And 6 samples have been done. The slices were cut by a vibrating-blade microtome and incubated with NB-BF (8 µM) in PBS bubbled with 5% CO2 for 30 min at 37 °C. Transferred the tissue slices to a glass bottomed dish (MatTek, 35 mm dish with 20 mm well) and imaged using OLYMPUSFV-1000 inverted fluorescence microscope with a 635 nm excitation and 655-755 nm emission. 4.11 Fluorescence imaging in vivo Tumor bearing mice were provided by Dr. Yao Kang. When implants grew up to about 0.5 cm in size, the mice were given a subcutaneous injection of NB-BF (200 µM/100 µL) at about 2 cm away from the tumour region, near the enterocoelia. After 30 min injection of NB-BF, the images of mice were obtained under a small animal imaging system (NightOWL II LB983) with an excitation laser of 630 nm and an emission filter of 700 nm. ASSOCIATED CONTENT Supplementary information is available: General information, additional spectroscopic, water solubility, pH stability, cell imaging data, and MTT results. Acknowledgements: This work was financially supported by the National Science Foundation of China (21421005, 21422601, 21576037, 21676047 and U1608222).


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Table of Contents


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Highly Selective Red-Emitting Fluorescent Probe for Imaging Cancer

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