Cyanine-based 1-amino-1-deoxyglucose as fluorescent probes for glucose transporter mediated bioimaging

Accepted Manuscript Cyanine-based 1-amino-1-deoxyglucose as fluorescent probes for glucose transporter mediated bioimaging Hu Xu, Xinyu Liu, Jinna Yang, Ran Liu, Taoli Li, Yunli Shi, Hongxia Zhao, Qingzhi Gao PII:

S0006-291X(16)30446-6

DOI:

10.1016/j.bbrc.2016.03.133

Reference:

YBBRC 35565

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 19 March 2016 Accepted Date: 27 March 2016

Please cite this article as: H. Xu, X. Liu, J. Yang, R. Liu, T. Li, Y. Shi, H. Zhao, Q. Gao, Cyaninebased 1-amino-1-deoxyglucose as fluorescent probes for glucose transporter mediated bioimaging, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.03.133. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Cyanine-based 1-amino-1-deoxyglucose as fluorescent probes for glucose

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transporter mediated bioimaging

Hu Xu,a Xinyu Liu,a Jinna Yang,b Ran Liu,a Taoli Li,a Yunli Shi,a Hongxia Zhao,*a and Qingzhi

Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation

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a

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Gao,*a

Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, P. R. China

Department of Biochemistry, Gudui BioPharma Technology Inc., 5 Lanyuan Road, Huayuan

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b

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Industrial Park, Tianjin 300384, P. R. China

Corresponding Author

* Tel: +86 135-1247-9137. Fax: +86 22-2789-2050. E-mail: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT Two novel cyanine-based 1-amino-1-deoxy-β-glucose conjugates (Glu-1N-Cy3 and Glu-1N-Cy5) were designed, synthesized and their fluorescence characteristics were studied. Both Glu-1N-Cy3

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and Glu-1N-Cy5 accumulate in living HT29 human colon cancer cells, which overexpress glucose transporters (GLUTs). The cellular uptake of the bioprobes was inhibited by natural GLUT substrate D-glucose and 2-deoxy-D-glucose. The GLUT specificity of the probes was validated with quercetin, which is both a permeant substrate via GLUTs and a high-affinity inhibitor of GLUT-mediated

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glucose transport. Competitive fluorometric assay for GLUT substrate cell uptake revealed that Glu-

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1N-Cy3 and Glu-1N-Cy5 are 5 and 10 times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. This study provides fundamental data supporting the potential of these two conjugates as new powerful tools for GLUT-mediated theranostics, in vitro and in vivo molecular

Keywords:

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bioimaging and drug R&D.

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Fluorescent probe, Glucose transporter, Fluorometric competition, Cyanine-based glycoconjugate

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1. Introduction Glucose is the main energy source in living systems and is transported into cells through glucose transporters (GLUTs) [1]. The imbalance of glucose homeostasis has been implicated in the pathogenesis of many diseases, including diabetes, cancer and obesity [2]. Therefore, the glucose

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molecular probes that can monitor cellular glucose transport have drawn intensive research attention as diagnostic tools; they have also been a focus for the discovery of novel therapeutic agents to treat

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metabolic diseases and cancer [3].

In the evaluation of glucose transport, radioligand displacement experiments have been widely

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adopted [4]. For instance, 3H or 14C labeled 2-deoxy-D-glucose (2-DG) and 3-O-methyl-D-Glucose (3-OMG) are usually being used for assessing cellular glucose uptake [5]. The most commonly used positron-emitting radio tracer, [18F]-2-fluoro-2-deoxyglucose (18FDG), is also a glucose analogue utilized extensively in cancer detection and therapeutic monitoring [6]. However, these radioisotope-

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based methods allow the cumulative measurement of cellular glucose at a fixed time point, but have limited applications in the real-time monitoring system for cellular glucose transport [7]. These limitations make them impractical or undesirable sometimes in cellular imaging and drug discovery

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research. In this context, fluorescence-based monitoring of cellular glucose uptake is thought to be a suitable alternative in laboratory research because of its distinct advantages in terms of sensitivity,

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selectivity, response time and applicability for bioimaging analysis in living systems [8–11]. The first fluorescent glucose derivative developed to probe the behaviour of glucose transport systems is 6-deoxy-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)-aminoglucose (6-NBDG) [12]. However, the use of this commercially available bioprobe by the biological research community was quite sparse according to the literature search result. This is presumably due to the fact that the conjugation at C-6 position would inhibit glucose phosphorylation, therefore, will limit its use for monitoring of cellular glucose metabolism.

In 1996, Matsuoka’s research group found that fluorophore 3

ACCEPTED MANUSCRIPT conjugation at the C-2 position of glucose to generate 2-NBDG would allow cellular phosphorylation and subsequent degradation to non-fluorescent products [13,14]. This fluorescent probe later became the most popular choice for various studies, particularly to image tumor glucose uptake and explore cellular metabolic functions associated with GLUT systems [14–18]. Nevertheless, many limitations

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also exist using 2-NBDG as a fluorescent probe, such as weak fluorescence intensity, high treatment dosage and noncompatibility in physiological conditions [7]. Therefore, developing novel glucose bioprobes is urgently needed. Recently, many fluorescent glucose bioprobes with features that are

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superior to NBDG were reported. Among them, the Cy3 and Cy5 fluorescent dyes have been the focus because of their compatibility with biological systems and tolerance to intense light sources

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[14]. Cy5.5-2DG developed by Gambhir has greater stability in the mouse model compared with 2NBDG and is useful in labelling cancer cells for cell trafficking and in vivo staining of tumours [19]. In 2011, Park’s group reported a series of Cy3-derived O-glycosides by coupling Cy3 fluorophore to the α-anomeric position of D-glucose with various linkers. Systematic and quantitative evaluation of

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these fluorescent glycosides led to the identification of one of the molecules, GB2-Cy3, which shows 10 times more sensitivity than 2-NBDG, as a GLUT-specific fluorescent glucose bioprobe [7].

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Inspired by these works, we designed and synthesized two novel cyanine-based 1-amino-1-deoxyβ-glucose conjugates, Glu-1N-Cy3 and Glu-1N-Cy5 (Scheme 1), through a direct amide coupling of

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1-amino-1-deoxy-D-glucose with cyanine fluorophores. As demonstrated in glycoprotein and glycopeptide studies, N-linked glycans behave much more stable against hydrolytic enzymes than that of O-glycomes under physiological and biochemical conditions [20]. With these two cyaninebased glycoconjugates, in this report, we shed light on their fluorescence characteristics, cytotoxic profiles and GLUT-dependent transportability from our preliminary evaluation studies.

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Scheme 1. Chemical structures of Glu-1N-Cy3 and Glu-1N-Cy5.

2. Materials and methods

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2.1. Materials

Cy3 and Cy5 were prepared according to the literature procedure [21]. The other starting material, 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosylamine, was prepared by the literature method [22]. All

purification.

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organic reagents were purchased from TCI Shanghai (Shanghai, China) and used without further

2.2. Synthesis of the fluorescent probes

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The preparation of Glu-1N-Cy3 and Glu-1N-Cy5 was accomplished by a two-step sequence from

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cyanine. The final products were completely characterized by 1H and 13C NMR spectroscopy, IR and HRMS analysis (see Supporting Information for detailed procedure and spectra data). 2.3. Cell cultures and cell uptake assay Human colon cancer cell line (HT29) was grown in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum in an atmosphere of 5% CO2 and 95% air at 37 °C. For the cell uptake assay of the probes, HT29 cells in 96-well black and clear bottom culture plate were grown to confluence, then washed twice with 200 µL glucose free culture medium and incubated for 60 minutes. After 5

ACCEPTED MANUSCRIPT washed with PBS for three times, the cells were treated with the PBS solution of the probes incubate for 90 minutes. At the end of the treatment, cells were washed five times with 2% dimethyl sulfoxide (DMSO) in PBS and subjected to fluorescence detection at the corresponding emission wavelength using Thermo Scientific Varioskan LUX multimode microplate reader. For quatitative purposes,

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cells were subsequently treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and subjected to MTT mediated cell viability assay in order to normalize the fluorescence levels according to the living cell numbers. Non-treated blank wells were used as the background,

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and the cell uptake of the bioprobes was calculated as the mean total fluorescence intensity subtracting background and normalized according to the mean living cells. Three independent

2.4. Fluorometric microplate assay

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experiments were performed in four to five duplicates and data are presented as mean ± S.E.

HT-29 cells (15,000 cells/well) were seeded in clear-bottomed 96-well microplates in triplicate.

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Cells were allowed to adhere overnight at 37 °C (for ~12 h) before performing uptake assays. After overnight incubation, the cells were carefully incubated with increasing concentrations of Glu-1NCy3 and Glu-1N-Cy5 (0–800 µM) for 120 min at 37 °C in a humidified atmosphere of 5% CO2. We

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stopped the reaction by adding a twofold volume of ice-cold PBS and the wells were washed again with ice-cold PBS 4 times. In order to normalize the fluorescence levels according to the living cell

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numbers, MTT solution (5 mg/mL) was prepared in PBS. It was added to the 96-well culture plates when the test of GC (glucose competitive assays) or the inhibition assays was finished. After a 4hour incubation at 37°C, the MTT medium was replaced with DMSO. The fluorescent signal (562 nm for Glu-1N-Cy3 and 665 nm for Glu-1N-Cy5) was measured using the Varioskan LUX Multimode Microplate Reader.

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ACCEPTED MANUSCRIPT Similarly, bioprobes uptake in HT-29 cells was evaluated by incubating cells (15,000 cells/well) with Glu-1N-Cy3 (100 µM) and Glu-1N-Cy5 (50 µM). The fluorescent signal was measured at specific time (10~180 min) using the Varioskan LUX Multimode Microplate Reader.

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2.5. Cytotoxicity assay The cellular growth inhibitory activity was determined using HT29 cell line. An amount of 6 x 103 cells per well were transferred to 96-well plates. After culturing for 24 h, the test compounds

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were added to triplicate wells at different concentrations and 0.1% DMSO for control. After 24 h of incubation, 20 µL of MTT solution (5 mg/mL) was added to each well, and the plate was shaken for

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1 min and then incubated for further 4 h at 37 °C. Cells were lysed by MTT lysis buffer (15% SDS, 0.015 M HCl) and uptake of MTT was measured at 570 nm absorbance using a multi-well–reading UV-Vis spectrometer. Experimental conditions were performed in five replicates (5 wells of the 96well plate per each experimental condition). All of the experiments were performed for three times.

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2.6. D-glucose and 2-DG mediated competitive inhibition assay

HT29 cells were grown in culture (as described in previous “cell culture” section) in clear-

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bottomed 96-well microplates at 15,000 cells/well and allowed to adhere overnight. After overnight incubation, the cells were carefully incubated with bioprobes (100 µM Glu-1N-Cy3 or 50 µM Glu-

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1N-Cy5) for 120 min in the absence or presence of increasing concentrations of D-glucose or 2deoxy-D-glucose (0-40 mM) for 120 min at 37 °C in a humidified atmosphere of 5% CO2. We stopped the reaction by adding a twofold volume of ice-cold PBS and the wells were washed with ice-cold PBS 4 times. For quantitative purposes, cells were subsequently subjected to MTT mediated cell viability assay in order to normalize the fluorescence levels according to the living cell numbers. 2.7. Quercetin mediated inhibition assay

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ACCEPTED MANUSCRIPT HT-29 cells (15,000 cells/well) were seeded in clear-bottomed 96-well microplates in triplicate. Cells were allowed to adhere overnight at 37°C (for ~24 h) before performing uptake assays. After overnight incubation, the cells were carefully incubated with bioprobes（100 µM Glu-1N-Cy3 or 50 µM Glu-1N-Cy5）for 90 min at 37 °C in a humidified atmosphere of 5% CO2. We stopped the

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reaction by adding a twofold volume of ice-cold PBS and the wells were washed with ice-cold PBS four times. The fluorescent signal (562 nm for Glu-1N-Cy3 and 665 nm for Glu-1N-Cy5) was measured using a Varioskan LUX Multimode Microplate Reader. Similarly, separate plates were

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prepared and treated with quercetin (20 µM) under standard culture conditions for 30 min. Then cells

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were treated with Glu-1N-Cy3 (100 µM) and Glu-1N-Cy5 (50 µM) for 90 min under standard culture conditions. For quantitative purposes, cells were subsequently subjected to MTT mediated cell viability assay in order to normalize the fluorescence levels according to the living cell numbers.

3. Results and discussion

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The synthesis of cyanine-based 1-amino-1-deoxy-β-glucose conjugates (Glu-1N-Cy3 and Glu1N-Cy5) was accomplished by a two-step sequence from cyanine (See supporting information). The intermediates were afforded in high yields by amide coupling of Cy3/Cy5 carboxylic acid with the β-

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D-glucopyranosylamine. Glu-1N-Cy3 and Glu-1N-Cy5 were obtained after deacetylation by EtONa

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and purification by flash column chromatography. With the fluorescent probes in hand, spectroscopic evaluation was conducted under physiological pH conditions (PBS, pH 7.4) by using a Thermo Scientific Varioskan LUX multimode microplate reader. In buffer, the excitation and emission maxima of Glu-1N-Cy3 are approximately 544 and 562 nm (Fig. 1A), whereas those of Glu-1N-Cy5 are approximately 644 and 665 nm, respectively (Fig. 1B). Their fluorescence properties lie in the visible spectral region, which has the potential for wide applications in the biological and clinical field. Furthermore, these two fluorescent probes have closely overlapping excitation and emission spectra. The energy emitted from Glu-1N-Cy3 may be 8

ACCEPTED MANUSCRIPT absorbed by Glu-1N-Cy5 when both are in close spatial proximity, which gives the potential to perform measurements on the single-molecule fluorescence resonance energy transfer (FRET) mediated spatial resolution studies.

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Then, whether the fluorescence intensity occurs in a concentration-dependent manner was evaluated. Fluorescence with different concentrations of Glu-1N-Cy3 and Glu-1N-Cy5 in PBS is shown in Fig. 1C and D. As expected, probes undergo a concentration-dependent change in their fluorescence intensity which demonstrates the linearity between the photoresponse of the probe and

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concentration of the analyte without fluorescence decay during dilution at physiological pH

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condition (ideal for competitive fluorometric assay). Moreover, Glu-1N-Cy5 shows about five times more greater fluorescent intensity than Glu-1N-Cy3 at the same concentration, this is due to the high

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quantum yield of Cy5 compared to Cy3 (Φ in PBS: Cy3 = 0.04; Cy5 =0.27) [23].

Fig. 1. Fluorescence spectra of the probes. (A) Fluorescent excitation and emission spectrum of Glu-1N-Cy3. (B) Fluorescent excitation and emission spectrum of Glu-1N-Cy5. (C) Fluorescence with different concentrations (109

ACCEPTED MANUSCRIPT 300 µM) of Glu-1N-Cy3 in PBS (λex = 544 nm, λem = 562 nm). (D) Fluorescence with different concentrations (10-200 µM) of Glu-1N-Cy5 in PBS (λex = 644 nm, λem = 665 nm).

After the spectroscopic evaluation, we proceeded to evaluate the applicability of our bioprobes in a bioassay system. To find the optimum concentrations for cellular uptake in competitive

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fluorometric assay experiments, HT29 human colon cancer cell line was selected as it is well established tumor type that over express GLUT1, 2, and 3 [24]. HT29 cells in 96-well black and clear bottom culture plate were treated with different concentrations of Glu-1N-Cy3 and Glu-1N-Cy5 (0–

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300 µM), respectively with 120 minutes incubation. After successive washing with PBS (containing 2% (v/v) DMSO), the fluorescent signal of dye labeled cells was measured using the Varioskan LUX

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Multimode Microplate Reader. As shown in Fig. 2A, the cellular uptake of the bioprobes was increased in a dose-dependent manner. The fluorescent microscopy cell imaging study also revealed the dose-response profile on both bioprobes (Fig. 2C).

In consideration of the high enough fluorescence intensities were observed in cells exposed to

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100 µM for Glu-1N-Cy3 and 50 µM for Glu-1N-Cy5 which reach 10 fold above of the equipment noise baseline level (high signal-to-noise ratio), therefore, these concentrations were considered as the optimum for the following cellular uptake experiments.

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With optimized concentrations for live-cell imaging, we used the Varioskan LUX Multimode

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Microplate Reader to measure the optimum incubation time required to achieve the maximum uptake of the bioprobes. Cellular uptake kinetic analysis of Glu-1N-Cy3 and Glu-1N-Cy5 in HT29 cells were performed using 100 µM of Glu-1N-Cy3 and 50 µM of Glu-1N-Cy5 for up to 180 min incubation (Fig. 2B). The amount of background fluorescence measured at time 0 were subtracted from values for each time point. The results show that both Glu-1N-Cy3 and Glu-1N-Cy5 uptake increase following incubation for 20 min or longer. The high fluorescence intensity (high signal-tonoise ratio) was observed after about 30 min and in a time-dependent manner and the fluorescence

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ACCEPTED MANUSCRIPT intensity reaches the maximum within approximately 120 min in PBS buffer (pH 7.4) at 25 °C for

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both bioprobes.

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Fig. 2. (A) Dose-dependent cell uptake of Glu-1N-Cy3 and Glu-1N-Cy5 in HT29 cells (10-300 µM, 2 h). (B) Uptake kinetics of Glu-1N-Cy3 and Glu-1N-Cy5 in HT29 cells (0-180 min). (C) Fluorescence microscopic imaging of HT29 cells stained by 100 µM of Glu-1N-Cy3 and by 50 µM of Glu-1N-Cy5. Excitation and emission for Glu-1N-Cy3: λex = 544 nm, λem = 562 nm, for Glu-1N-Cy5: λex = 644 nm, λem = 665 nm. Objective lens: 40x.

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As small molecule fluorescent probe that may potentially be used for extensive biological examinations, e.g. we assume that our probes would be effective and useful to explore cellular

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metabolic functions associated with GLUT systems. Owing to these reasons, the probes should not cause toxicities to the biological systems. Hence, the toxicity assessment using HT29 cells treated with Glu-1N-Cy3 (100 µM) and Glu-1N-Cy5 (50 µM) was performed by MTT mediated viability measurement. From the MTT assay, we observed that around 82.3% and 98.9% of cell viability can be maintained after 24 hours of incubation respectively at the tested concentrations (see the Supporting Information). With this result, we could conclude that under our standard bioimaging analysis conditions (≤100 µM and 120 min), the probes do not cause detectable toxicity to adversely affect cell viability. 11

ACCEPTED MANUSCRIPT Further experiments were aimed at determining whether the uptake of Glu-1N-Cy3 and Glu-1NCy5 was mediated by GLUTs. If the cellular uptake of certain glucose analogues depends on the concentration of D-glucose or 2-DG, the particular glucose analogues would enter the cell through GLUT transport system [22]. We performed competition analysis with increasing concentrations of

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D-glucose and 2-DG respectively with Glu-1N-Cy3 and Glu-1N-Cy5 in HT29 cells. As shown in Fig. 3A and B, the cellular uptake of our bioprobes were dose dependently reduced upon competition with D-glucose and 2-DG, indicating that the cellular uptake of the bioprobes at least regulated via

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GLUT mediated transport systems. To further confirm the GLUT transportability of the bioprobes, we determined the dependency between cellular uptake of the probes in HT29 and GLUTs by using

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quercetin as a glucose transporter inhibitor. Quercetin has been demonstrated to be an effective inhibitor of GLUT1, 2, and 5, as well as of sodium dependent vitamin C transporter SVCT1 [26-29]. Based on our previous studies, 20 µM of quercetin can be used to effectively inhibit GLUT mediated substrate transport without potential toxicity or viability interference may cause by the inhibitor [30].

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Experiment results depicted in Fig. 3A and B proved that quercetin treatment obviously decreased the cellular uptake of the bioprobes in HT29 cells, indicating that the uptake of Glu-1N-Cy3 and Glu-1N-Cy5 is mainly mediated by GLUTs. Compared to Glu-1N-Cy3, Glu-1N-Cy5 was found to

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be more sensitive to this inhibitory effect, that the cell uptake decrease caused by 20 µM of quercetin

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for Glu-1N-Cy5 was about 60% while this percentage change for Glu-1N-Cy3 was about 40%. Fig. 3C shows the fluorescence microscopic imaging of HT29 cells. High concentration of the natural GLUT substrate, 2-deoxy-D-glucose (40 mM) effectively diminished the fluorescence intensity of the cells by competing with the bioprobes (100 µM Glu-1N-Cy3 and 50 µM of Glu-1NCy5).

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Fig. 3. (A) GLUT inhibitor quercetin, D-glucose and 2-DG competitive inhibition assay in Glu-1N-Cy3 (100 µM) labeled HT29 cells. (B) GLUT inhibitor quercetin, D-glucose and 2-DG competitive inhibition assay in Glu-1NCy5 (50 µM) labeled HT29 cells. Quer: Quercetin. Glu: D-Glucose. 2DG: 2-Deoxy-D-glucose. (C) Fluorescence microscopic imaging of Glu-1N-Cy3 and Glu-1N-Cy5 labeled HT29 cells from competitive inhibition assay. Excitation and emission for Glu-1N-Cy3: λex = 544 nm, λem = 562 nm, for Glu-1N-Cy5: λex = 644 nm, λem = 665 nm. Objective lens: 40x.

Having confirmed that the uptake of Glu-1N-Cy3 and Glu-1N-Cy5 was mediated by GLUTs, we compared them with the first generation fluorescent glucose bioprobe, namely, 2-NBDG. Based on

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previous reports, 2-NBDG has been proven to be a substrate of GLUTs [13,17,31]. From our comparison study, approximately 60% of 2-NBDG (500 µM) uptake is competitively inhibited in the

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presence of 40 mM 2-DG (Fig. S11). However, low concentration of our bioprobes, Glu-1N-Cy3 (100 µM, Fig. 3A) and Glu-1N-Cy5 (50 µM, Fig. 3B), can achieve the same effect. In addition, the cellular uptake of 2- NBDG in normal media (containing 10 mM D-glucose) is extremely low and can hardly be detected through fluorescence-based imaging methods, which makes it quite limited in biologically significant environments [16,25]. In contrast to the results with 2-NBDG, our fluorescent bioprobes can be applied in a bioassay system without glucose starvation because they are sensitively detectable even under high concentration (40 mM) of 2-DG. Thus, our bioprobes 13

ACCEPTED MANUSCRIPT showed superior properties as a glucose-uptake tracer compared with 2-NBDG. More specifically, Glu-1N-Cy3 and Glu-1N-Cy5 are about 5 times and 10 times more sensitive than 2-NBDG is in competitive fluorometric assay.

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4. Conclusion

GLUT-mediated molecular fluorescent bioimaging probes are potentially useful as in vitro and in

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vivo tools to evaluate intracellular glucose metabolic kinetics, cell- and tissue-based glucose homeostasis (e.g., brain and neuron energy uptake), Warburg effect-mediated cancer detection and

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therapy as well as noninvasive glucose sensing in diabetes management and diagnosis. In this study, we developed two novel fluorescent glucose bioprobes, Glu-1N-Cy3 and Glu-1NCy5, as useful tools for GLUT-mediated molecular sensing and bioimaging. Compared with the first-generation glucose bioprobe 2-NBDG, the current probes have high sensitivity and non-toxicity.

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The fluorescence excitation and emission properties of the developed glucose bioprobes lie in the visible spectral region, which has the potential for broad range of applications in the biological and clinical field. In this study, we demonstrated that our probes could be utilized for the real-time and

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quantitative monitoring of cellular glucose uptake in living cells. Both Glu-1N-Cy3 and Glu-1N-

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Cy5 are GLUT-specific and can be potentially utilized in microplate-based fluorometric assay for drug screening of Warburg effect-targeted anti-tumour agents. As opposed to the traditional glucose uptake assay with radioisotope-labelled probe, Glu-1N-Cy3 and Glu-1N-Cy5 allow a sufficiently sensitive real-time monitoring of glucose transport in GLUT-overexpressed living cells, which provides a powerful tool for chemical biology and biomedical science. Furthermore, the combination of Glu-1N-Cy3 and Glu-1N-Cy5 also has potential to perform measurements on the single-molecule FRET-mediated spatial resolution studies in biological research. More applications and evaluation

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Acknowledgements

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This research was supported by Grants from the Tianjin Municipal Applied Basic and Key Research Scheme of China (11JCYBJC14400, 12ZCDZSY11500, 13JCZD27500), and by the

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Project of National Basic Research (973) Program of China (2015CB856500).

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Research Highlights




Cy-3 and Cy-5 derived new bioprobes were prepared for glucose transporter mediated

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bioimaging. The probes accumulate in living HT29 human colon cancer cells.




The cellular uptake of the probes was inhibited by natural GLUT substrates and inhibitor.




The probes are 5-10 times more sensitive than 2-NBDG in competitive fluorometric assay

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