A Novel Estrogen Receptor Î±-Targeted Near-Infrared Fluorescent

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A novel estrogen receptor #-targeted near-infrared fluorescent probe for in vivo detection of breast tumor Chu Tang, Yang Du, Qian Liang, Zhen Cheng, and Jie Tian Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00684 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 6, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Molecular Pharmaceutics

A novel estrogen receptor α-targeted near-infrared fluorescent probe for in vivo detection of breast tumor Chu Tang1,2‡, Yang Du2,3,4‡, Qian Liang 2, Zhen Cheng5*, Jie Tian1,2,3,4*

1

Engineering Research Center of Molecular and Neuro Imaging, Ministry of Education, School

of Life Science and Technology, Xidian University, Xi’an, Shaanxi 710126, China; 2

CAS Key Laboratory of Molecular Imaging, The State Key Laboratory of Management and

Control for Complex Systems, Institute of Automation, Chinese Academy of Sciences, Beijing 100190, China; 3

Beijing Key Laboratory of Molecular Imaging, Beijing, 100190, China

4

University of Chinese Academy of Sciences, Beijing, 100080, China

5

Molecular Imaging Program at Stanford (MIPS), Department of Radiology, and Bio-X Program,

Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, California, 94305-5344; ‡

These two authors contributed equally to this work

*Corresponding author: Jie Tian, E-mails: [email protected], [email protected]. Phone.: 8610 62527995, Fax: 86 10 62527995, Website: http://www.3dmed.net; Zhen Cheng, E-mail: [email protected]. Phone: 650-723-7866, Fax: 650-736-7925.

1

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: The ability to detect breast cancer early in its progression is essential to improve patient survival and quality of life. The noninvasive and dynamic in vivo imaging and functional assessments of estrogen receptor-alpha (ERα), which is commonly expressed at high levels in breast cancer, are important for effective diagnosis and treatment. Hence, the development of a specific ERα-targeted probe is a major research goal. To that end, in the present study we created a novel near-infrared (NIR) fluorescent probe, IRDye800CW-E2, for the targeted in vivo ERα imaging in breast tumor-bearing mice. IRDye800CW-E2 consisted a cyanine dye IRDye800CW as the NIR fluorophore and the E2 analogue ethinyl estradiol amine as an ERα targeting ligand. The ethinyl estradiol amine was initially labeled with fluorescein isothiocyanate (FITC) to evaluate the binding specificity to human breast tumor cells in vitro. Flow chamber and in vitro confocal laser endomicroscopy imaging experiments demonstrated FITC-E2 was specifically taken up by MCF-7 cells. Furthermore, in vivo NIR fluorescence imaging revealed the ability of IRDye800CW-E2 to rapidly target tumors and to achieve good contrast between tumors and background signal 4 - 48 h post-injection. The fluorescent signal of IRDye800CW-E2 in tumors was successfully blocked by the coinjection of the endogenous ERα ligand 17β-estradiol (E2) and the probe. Ex vivo fluorescent imaging further confirmed high uptake of the probe by tumors. These results indicated that IRDye800CW-E2 has great potential as an ERα-targeted imaging probe for the early breast tumor detection and has potential for clinical translation.

KEY WORDS: breast cancer, NIR fluorescent probe, ERα-targeted imaging, in vivo detection

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INTRODUCTION Estrogen receptor-alpha (ERα) is a nuclear hormone receptor responsible for regulating physiological effects of signaling associated with endogenous estrogens, such as 17β-estradiol (E2), in diverse target tissues.1-3 Although the binding of ERα to its ligand E2 plays a critical role in female reproductive organs, aberrant ERα signaling can mediate a hormone imbalance and is associated with several cancers,4,5 especially breast cancer.6,7 Nearly 75% of patients with breast cancer demonstrate abnormally high expression of ERα.8 Therefore, ERα has emerged as a key target for breast tumor therapy, and several ERα ligands have been developed as antagonists against ERα-positive [ERα (+)] breast cancer.9-11 Despite improvements in overall survival for patients who receive endocrinotherapy, recurrence and metastasis are serious problems.12,13 ERα-targeted imaging can be used to identify suitable treatment regimens, and hence can improve patient outcomes. Currently, the primary molecular imaging techniques for ERα are single photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI) with the application of radiolabeling or metal-chelating selective estrogen receptor modulators.14-17 These methods offer excellent potential for quantitative imaging to determine the levels of ERα expression18,19 but are not routinely used for breast cancer detection and diagnosis owing to the poor specificity and high costs. Optical imaging is a promising molecular imaging technology for cancer owing to its high sensitivity, high specificity, safety, low cost,20-22 and ease compared to nuclear imaging and magnetic resonance imaging modalities.23 Recently, a few ERα-targeted fluorescent probes have been developed, but they are mainly used to determine the binding affinity of ligands or the localization of ER proteins in cells;24-26 the in vivo targeted imaging of ERα for breast tumor detection has not been performed. In the present study, we developed an ERα-targeted molecular probe for ERα (+) breast tumor-targeted imaging in vivo. Based on an analysis of the crystal structure of the ERα-ligand binding domain (ERα-LBD) complexed with E2, the A-ring phenolic 3



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hydroxyl and D-ring hydroxyl of E2 form hydrogen bonds with helix 3/E353 and helix 11/H524, which are the key elements for maintaining high-affinity ligand binding.1 Hence, the fluorophores cannot be directly conjugated to the hydroxyl groups of E2 but are coupled by the linking group. Herein, an E2 derivative (namely, ethinyl estradiol amine, 4) with an amino linking group was prepared by click chemistry. To quantitatively analyze the cellular uptake of 4 by ERα-overexpressing breast tumors at the cellular level, due to current detection equipment limitations, 4 was labeled with fluorescein isothiocyanate (FITC) to be used in flow cytometry and the confocal experiments. Fluorophores with emission in the near-infrared (NIR) region possess less absorption and scatter from tissues much more efficiently than fluorophores based on visible light;27,28 thus, for in vivo imaging using MCF-7 [ERα (+)] tumor xenografts, 4 was labeled with the NIR dye IRDye800CW to yield IRDye800CW-E2. Our results demonstrated that IRDye800CW-E2 is a sensitive and efficient NIR fluorescent probe for ERα expression visualization in ERα (+) breast tumors in vivo with high clinical translation potential. MATERIALS AND METHODS Materials. All reagents were obtained from Aldrich (St. Louis, MO, USA), Acros (Geel, Belgium), Aladdin Reagent (Shanghai, China), and Alfa-Aesar (Haverhill, MA, USA).

No

additional

purification

was

performed.

Analytical

thin-layer

chromatography (TLC) was used for reaction monitoring, with visualization performed via UV light (254 nm). A Bruker Biospin AV400 (400 MHz, 1H NMR; 100 MHz, 13C NMR) instrument was utilized to measure the 1H NMR and 13C NMR spectra. Chemical shifts (in ppM) were in reference either to tetramethylsilane or the solvent. High-performance liquid chromatography (HPLC) allowed for confirmation of >95% purity of all compounds used in a biological context. Cell lines. MCF-7 cells (ATCC, USA) and normal breast epithelial MCF-10A cells (Chinese Academy of Sciences) were grown in a 37 °C 5% CO2 incubator in Dulbecco’s Minimum Essential Medium (DMEM, Hyclone, Thermo Scientific) 4


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supplemented with 10 % fetal bovine serum (FBS, Hyclone, Thermo Scientific) as well as penicillin and streptomycin. In vivo tumor model. The Institutional Animal Care and Use Committee of Peking University approved this study (Permit No: 2011-0039). For in vivo tumor assays, female BALB/c mice (6-week-old, 15-18 g; Beijing Vital River Laboratory Animal Technology Co., Ltd) received axillary subcutaneous transplants of MCF-7 cells. Synthesis of ethinyl estradiol amine (4). NaN3 (90 mg, 1.38 mmol) was mixed into a solution of 3-chloropropylamine hydrochloride 2 (60 mg, 0.46 mmol) in H2O2 (2 mL). This solution was brought to 80 °C for 12 h, cooled, basified by KOH (1 mol/L) to pH 9, and diethyl ether was employed for extraction (3 × 10 mL). Na2SO4 was utilized for organic layer drying, and this layer was concentrated with a flow of nitrogen to obtain azide 3 as a white oil. The unpurified residue was used in the following reaction. A mixture of azide 3 (10 mg, 0.10 mmol) and ethinyl estradiol 1 (30 mg, 0.10 mmol) in tBuOH (500 µL) was supplemented with H2O (500 µL), ascorbic acid (2 mg, 11 µmol), and CuSO4⋅5H2O (3 mg, 12 µmol), and was stirred at 25 ºC for 12 h. This crude mixture was diluted using ethyl acetate (40 mL) and 4:1 saturated NH4Cl/NH4OH (40 mL). The organic layer was separated and washed with 4:1 saturated NH4Cl/NH4OH (3 × 40 mL), dried, and concentrated under a vacuum to yield 24 mg (61.7%) of ethinyl estradiol amine 4 as a white solid. 1H NMR (400 MHz, CD3OD): δ 7.84 (s, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.50 (d, J = 8.4 Hz, 1H), 6.47 (s, 1H), 4.44-4.49 (m, 2H), 2.71-2.78 (m, 3H), 2.43-2.501 (m, 1H), 2.072.12 (m, 4H), 1.80-2.004 (m, 43H), 1.51-1.64 (m, 3H), 1.35-1.43 (m, 2H), 1.24-1.32 (m, 1H), 1.04 (s, 3H), 0.65-0.70 (m, 1H); 13C NMR (100 MHz, CD3OD): δ 154.53, 154.12, 137.42, 131.03, 125.75, 122.76, 114.70, 112.32, 81.82, 48.20, 46.96, 43.50, 39.64, 37.08, 32.97, 29.33, 27.33, 26.13, 23.25, 13.56. MS (ESI): m/z 317.26 ([M+H]+). Synthesis of FITC-E2 conjugate (5). A FITC solution (0.8 mg, 2.1 µmol) in dry DMSO (2 mL), 4 (0.9 mg, 3.1 µmol) and Et3N (50 µL) were added and stirred in the dark at 37 ºC for 12 h. Subsequently, a C-18 HPLC column (250 mm × 10 mm) was 5



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used to purify this crude product using gradient elution with CH3CN and H2O as solvents A and B, respectively. The purified FITC-E2 was obtained as a yellow solid with 81.9% yield and over 98% purity by analytical HPLC. MS (ESI): m/z 699.78 ([M]+). Synthesis

of

IRDye800CW-E2

conjugate

(6).

To

a

solution

of

IRDye800CW-NHS (0.5 mg, 0.43 µmol) in dry DMSO (2 mL), 4 (0.2 mg, 0.64 µmol) and Et3N (30 µL) were added, stirring protected from light at 37 ºC for 24 h, followed by trifluoroacetic acid (50 µL) quenching. Subsequently, a C-18 HPLC column (250 mm × 10 mm) was used for crude product purification using gradient elution with CH3CN (0.1% TFA) and H2O as solvents A and B, respectively. The purified IRDye800CW-E2 was obtained as a yellow solid with 81.9% yield and over 98% purity by analytical HPLC. MS (ESI): m/z 1295.418 ([M-H]-). In Vitro Cellular Uptake Assays. In order to quantitatively assess breast cancer cell FITC-E2 uptake, MCF-7 and MCF-10A cells received several concentrations of FITC-E2 or FITC for 3 h, followed by washing with ice-cold PBS. Finally, the cells were dispersed in ice-cold PBS and used for flow cytometry, cellular uptake, and fluorescence measurements. In vitro Confocal Microscopy Images. 4 was labeled with FITC to yield probe FITC-E2, which was used for the flow chamber and confocal microscopic experiments. MCF-7 cells grown in six-well plates were treated with 0.5 µM FITC-E2 or 50-fold excess E2 was added as blocking agent for 3 h. The cells were then washed using ice-cold PBS prior to rhodamine phalloidin and DAPI staining. The fluorescence signals were then recorded via confocal laser scanning microscopy (Leica Microsystems). In vivo Confocal Laser Endomicroscopy (CLE) Imaging. Cellvizio was used for CLE imaging with dual band excitation wavelengths of 488 and 660 nm. To conduct in vivo CLE imaging, FITC-E2 was injected into mice bearing MCF-7 tumors. Fluorescence images were acquired before injection and at 2, 4, 6, 12, 24, and 48 h later with the UltraMiniO microprobe. Moreover, 12 h after the probe injection, Evans 6


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blue was injected via the tail vein to the blood vessel for 15 min, and then the confocal probe was directly inserted into the tumor by a superficial skin incision to scan the signal with a S-1500 microprobe. Estrogen Receptor Binding Affinity. A fluorometric binding assay was employed to assess binding affinities, as described in past studies29,30. Relative binding affinity (RBA) values were used to report these results, based on an E2 RBA of 100%. Each measurement was performed in triplicate. Molecular modeling. The Protein Data Bank was queried to obtain the ERα crystal structure (PDB: 2P15)31, and water molecules were removed. The crystallographic coordinates of 6 was prepared using Biochemoffice. AutoDockTools (ADT) were used for ligand and protein performance, with AutoDock (version 4.2) used for all molecular docking studies.32,33 PyMOL (Erwin Schrödinger) was used to prepare figures. In vivo Fluorescent Imaging. Fluorescent imaging was conducted to assess the tumor-targeting effects and the in vivo IRDye800CW-E2 probe biodistribution. For fluorescence imaging studies, 10 nmol IRDye800CW-E2 was intravenously injected into the tumor-bearing mice (n = 3); images were acquired prior to injection and at 2, 4, 6, 12, 24, and 48 h after injection via IVIS spectrum imaging system. For blocking studies, tumor-bearing mice (n = 3) were coinjected with 10 nmol IRDye800CW-E2 and 100 nmol endogenous E2. Regions of interest (ROI) of the tumors and adjacent normal tissues were measured, and then the ratios of tumor to normal tissue fluorescence signals were determined. After 48 of imaging, dissection of the tumor and major organs was performed to facilitate ex vivo imaging acquisition. ROI analyses of the average fluorescence signals were also performed with the IVIS spectrum imaging system. Statistics. Data are given as means ± SD of triplicate experiments. One-way ANOVAs followed by Tukey test were used to compare differences between groups, while two-tailed Student’s t tests were used to compare two groups. *P < 0.05 was the threshold of significance. 7



RESULTS AND DISCUSSION Chemical Synthesis. FITC-E2 5 and IRDye800CW-E2 6 were synthesized by conjugating FITC or the NHS-activated ester of IRDye800CW to the amino of ethinyl estradiol amine 4 in the presence of triethylamine, as shown in Scheme 1. Key intermediate 4 was prepared by utilizing the azide-alkyne click chemistry reaction of ethinyl estradiol 1 and azide 3 in the presence of the Cu(I) catalyst, and organic azide 3 was synthesized by reacting 3-chloropropylamine 2 with NaN3 at 80 °C in H2O for 12 h.

Scheme 1. Synthesis of FITC-E2 and IRDye800CW-E2. Reagents and conditions: (a) NaN3, H2O, 80 ºC, 12 h; (b) ascorbic acid, CuSO4·5H2O, tBuOH/H2O, 25 ºC, 12 h; (c) FITC, TEA, DMSO, 37 ºC, 12 h; (d) IRDye 800CW-NHS, TEA, DMSO, 37 ºC, 12 h.

Cellular uptake study. Flow cytometry was used for the quantitative analysis of cellular uptake and fluorescence enhancement by 4. Flow chamber experiments showed that the percentage of cellular uptake and the mean fluorescence intensity of FITC-E2 by MCF-7 cells were significantly higher than free FITC (Fig. 1A, 1B), but there were no statistically significant difference between FITC-E2 and FITC in MCF-10A (Fig. 1C, 1D). These results indicated that 4 possessed active breast tumor-targeting effects, and FITC-E2 can specifically bind to breast cancer cells. 8


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Figure 1. Examination of the cellular uptake of FITC-E2 by flow cytometry. (A) Quantitative analysis of FITC-E2 and FITC uptake in MCF-7 cells by flow cytometry; (B) Fluorescence intensity of FITC-E2 and FITC in MCF-7 cells; (C) Quantitative FITC-E2 uptake analysis in MCF-10A cells; (D) Fluorescence intensity of FITC-E2 in MCF-10A cells. *P < 0.05, **P < 0.01.

In vitro confocal imaging. To further study the specific intracellular uptake of FITC-E2, confocal laser microscopy was applied to visualize and track FITC-E2 within MCF-7 tumor cells. After exposure of tumor cells to FITC-E2 for 4 h, the actin cytoskeleton and nuclei of MCF-7 cells were stained with rhodamine phalloidin and DAPI. FITC-E2 was taken up by MCF-7 cells (Fig. 2), as seen in the flow cytometry results. The cellular uptake of FITC-E2 can be effectively blocked by co-incubation with E2 (Fig. 2B), which further confirmed the targeting specificity of FITC-E2 for MCF-7 cells.

9



Figure 2. Targeting specificity of FITC-E2 in vitro. Rhodamine phalloidin (red) and DAPI (blue) were used to stain the actin cytoskeleton and nuclei, respectively. (A) MCF-7 cells incubated in FITC-E2; (B) MCF-7 cells co-incubated in FITC-E2 with 50-fold excess E2. Scale bar represents 10 µm.

In vivo confocal laser endomicroscopy (CLE) imaging. To further verify the specific uptake of FITC-E2 by breast tumor tissues, probe-based confocal laser endomicroscopy (pCLE), a recently developed approach offering real-time, in vivo and very high magnification images, was used to evaluate MCF-7 tumor xenografts (Fig. 3 and 4). After the intravenous injection of FITC-E2, fluorescence signals can be detected at the tumor region as early as 2 h, and the fluorescence signal can be detected for 48 h post-injection (Fig. 3A). Most FITC-E2 was rapidly cleared from adjacent healthy tissues within 4 h (Fig. S1, Fig. 3B). At 12 h, there was a sufficiently high tumor/normal tissue fluorescence signal ratio to clearly discern the tumor region, and the probe FITC-E2 could be observed mainly in the tumor cytoplasm (Fig. 4A). In contrast, there was no obvious signal in adjacent healthy tissues (Fig. 4B). These results indicated that the FITC-E2 can efficiently traffic via blood vessels to target tumor tissues and possessed in vivo targeting capability.

10


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Figure 3. Probe-based confocal laser endomicroscopy fluorescent images for the UltraMiniO microprobe; green fluorescence is the FITC-E2 signal. (A) Tumor tissue; (B) adjacent normal tissues. Scale bar represents 20 µm.

Figure 4. The probe-based confocal laser endomicroscopy fluorescent images of tumor with S-1500 microprobe at 12 h post-injection of probe FITC-E2. The green signal is the fluorescence of FITC-E2, and blood vessel was stained with Evans blue shown in red. (A) Tumor tissue; (B) adjacent normal tissues. Scale bar = 50 µm.

The Binding Affinity of the NIR Probe IRDye800CW-E2. These results suggested the possible application of 4 for in vivo fluorescence imaging. To fully examine the in vivo tumor targeting effects and biodistribution of 4, conjugation to the IRDye800CW was conducted yielding IRDye800CW-E2 as a NIR probe. Currently, a major challenge in the development of steroid (steroidal)-based ER-targeted fluorescent probes or drug delivery systems is the maintenance of a high affinity for ER and subtype selectivity. Most steroidal-conjugates show extremely low 11



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ER binding affinity and weak subtype selectivity because conjugating a functional group to steroids often decreases the ER binding affinity,34-36 and can even lead to the loss of affinity for small linkers to steroid molecules.37 Katzenellenbogen et al. proposed that the relative binding affinity (RBA) for such ER-targeted conjugates should be at least 1% of the value for E2 (RBA = 100%).38 Therefore, we first used a competitive fluorometric receptor binding assay to test the ERα and ERβ binding affinity of IRDye800CW-E2 probe (Table 1). The RBA is used to present these affinities, where E2 was set as 100%. Our results showed that 4 had good ERα binding affinity, with ERα and ERβ RBA values of 4.44 and 2.53, respectively, and an overall ERβ/ERα selectivity of 1.7 (Table 1). More interestingly, conjugating the IRDye800CW moiety to 4 not only obviously increased the ERα binding affinity of the probe IRDye800CW-E2, but also exhibited higher subtype selectivity for ERα (RBA values were 8.06 for ERα and 0.53 for ERβ, α/β was as high as 15.2, Table 1). Table 1. ERα and ERβ relative binding affinitiesa RBAa (%)

Kib (nM)

Compound

a

ERα

ERβ

α/β

ERα

ERβ

α/β

4

4.44 ± 0.18

2.53 ± 0.32

1.7

69.82

134.39

1.9

IRDye800CW-E2

8.06 ± 0.32

0.53 ± 0.05

15.2

38.46

641.51

16.6

Relative binding affinity (RBA) values were calculated based on the results of a competitive

fluorometric receptor binding assay and are expressed as IC50estradiol/IC50compound × 100 ± the standard deviation (RBA, estradiol = 100%). bKi values of each compound for each receptor were obtained for the RBA values by formula Ki = (100/RBA) × Kd. The estradiol Kd values are 3.1 nM on ERα and 3.4 nM on ERβ.29

Molecular Docking analysis of probe binding. To understand how the probe fit into the ERα ligand pocket, we performed a molecular docking study with IRDye800CW-E2 in ERα (PDB: 2P15) to examine the binding mode.31 Consistent with E2 (Fig. 5A), IRDye800CW-E2 also can bind in an optimal conformation in the ERα active pocket (Fig. 5B), and phenolic hydroxyl of the steroidal ring forms critical 12


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hydrogen bond contacts with helix 3/E353 and the fluorophore unit also exhibits a key interaction with helix 11/H524 (Fig. 5C); in addition, the IRDye800CW group forms additional hydrogen bonds with A340 residue (Fig. 5C). This may explain why conjugating the fluorophore IRDye800CW to E2 does not affect the ERα binding affinity.

Figure 5. Molecular modeling of IRDye800CW-E2 bound to ERα, and E2 comparisons. (A) ERα LBD in complex with E2 crystal structure (1ERE).1 Hydrogen bonds are present between E2 and E353, R394, H524. (B) Computer modeling of the complex of IRDye800CW-E2 bound to ERα with the conserved H-bond to A340, T347, E353 and H524. (C) IRDye800CW-E2 binding deep into the ERα active pocket.

In vivo NIR fluorescent imaging of IRDye800CW-E2. After the intravenous injection of 10 nmol IRDye800CW-E2 in nude mice that had received MCF-7 human breast tumor xenografts, fluorescence images were dynamically monitored at various time points. The probe IRDye800CW-E2 rapidly targeted tumor areas; fluorescence signals were detected as early as 4 h, reached a peak at around 12 h, and persisted for 48 h post-injection. (Fig. 6A). The tumor-to-background ratios (TBR) were 1.78 ± 0.20 at 2 h, 3.34 ± 0.46 at 12 h, and 2.43 ± 0.19 at 48 h (Fig. 6C). To further determine the specificity of IRDye800CW-E2 targeting to ERα, a blocking study was also performed by the co-injection of IRDye800CW-E2 in combination with the ERα 13



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ligand E2. As shown in Fig. 6B, the tumor uptake of IRDye800CW-E2 decreased dramatically after blocking compared to that of the imaging group. Moreover, the TBR in the blocking group was also lower than that in the imaging group, confirming the specificity of IRDye800CW-E2 for ERα. After the in vivo observations, the ex vivo tumors and major organ imaging was conducted. As shown in Fig. 6D, a higher fluorescence signal was evident within the tumor after injection with the IRDye800CW-E2 probe in the imaging group than the signal in the blocking group. The average radiant efficiency of tumor fluorescence signals in the blocking group as 64.2% lower than that in imaging group (Fig. 6E). The

efficient blocking

study

further confirmed

the

specific

binding

of

IRDye800CW-E2 to ERα. The fluorescence signal for IRDye800CW-E2 was also detected in the liver and kidney, indicating the elimination of IRDye800CW-E2 mainly through the kidney and, to a lesser extent, through the liver.

Figure 6. In vivo fluorescence images and the biodistribution of IRDye800CW-E2 in MCF-7 tumor-bearing nude mice. (A) Representative mouse NIR fluorescence images before injection and at 2, 4, 6, 12, 24, and 48 h after the injection of 10 nmol IRDye800CW-E2; (B) Representative NIR fluorescence images of mice with excess E2 blocking; (C) Region of interest analysis of tumor to normal tissue fluorescence signal ratios of IRDye800CW-E2 in the imaging and blocking 14


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groups; (D) Representative ex vivo organ NIR fluorescence images (Labels: T, tumor; H, heart; L, liver; S, spleen; L, lung; K, kidney; I, intestines) at 48 h post-injection of 10 nmol IRDye800CW-E2 with (blocking group) or without (imaging group) 100 nmol E2; (E) Quantitative analysis of fluorescence intensity of dissected organs. *P < 0.05.

CONCLUSION In the present study, we have succeeded in the design and synthesis of the novel ERα-targeted NIR fluorescent probe IRDye800CW-E2 for targeted ERα (+) breast tumor imaging in vivo. This is, to the best of our knowledge, the only application of IRDye800CW-E2 as a fluorescent probe for the in vivo imaging of ERα for breast tumor detection to date. The IRDye800CW-E2 probe exhibited good ERα binding affinity. For in vivo imaging studies, IRDye800CW-E2 was able to efficiently target ERα with high imaging sensitivity and specificity and good tumor-to-background ratios in MCF-7 xenograft tumors. Our results indicated that IRDye800CW-E2 shows potential to efficiently visualize ERα levels in breast tumors and enable the early detection and treatment of breast cancer, offering potential for clinical translation.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Cartoon displays how to select adjacent normal tissue and 1H NMR and

13

C

NMR spectral information for compound 4 (PDF)

AUTHOR INFORMATION Corresponding Authors Jie Tian, E-mails: [email protected], [email protected]. Phone.: 8610 62527995, Fax: 86 10 62527995, Website: http://www.3dmed.net; Zhen

Cheng,

E-mail:

[email protected].

Phone:

15


650-723-7866,

Fax:


Page 16 of 22

650-736-7925. Notes The authors declare no conflicts of interest.

ACKNOWLEDGEMENTS This paper is supported by Ministry of Science and Technology of China under Grant No. 2017YFA0205200, 2017YFC1309100, 2016YFC0102000, the National Natural Science Foundation of China under Grant No. 81470083, 81527805, 61231004, 81601548, 81772011, 81701771, the International Innovation Team of CAS under Grant No. 20140491524, Beijing Municipal Science & Technology Commission No. Z161100002616022, China Postdoctoral Science Foundation funded project (2017M613084),

Shaanxi

Postdoctoral

Science

Foundation

funded

project

(2017BSHEDZZ91).

ABBREVIATIONS ERα, estrogen receptor-alpha; E2, 17β-estradiol; NIR, near-infrared; ERα-LBD, ERα-ligand binding domain; TLC, analytical thin-layer chromatography; TEA, triethylamine;

HPLC,

high-performance

liquid

chromatography;

MS,

mass

spectrometry; DMEM, dulbecco minimum essential medium; FBS, fetal bovine serum; RBA,

relative

binding

affinity;

PDB,

protein

data

bank;

FITC,

fluorescein-5-isothiocyanate; DAPI, 4',6-diamidino-2-phenylindole; CLE, confocal laser endomicroscopy; ROI, regions of interest.

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Table of Contents

A novel estrogen receptor α-targeted near-infrared fluorescent probe for in vivo detection of breast tumor Chu Tang, Yang Du, Qian Liang, Zhen Cheng, Jie Tian

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A Novel Estrogen Receptor Î±-Targeted Near-Infrared Fluorescent

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