A unique red-emitting two-photon fluorescent probe with tumor-specificity for imaging in living cells and tissues

Author’s Accepted Manuscript A unique red-emitting two-photon fluorescent probe with tumor-specificity for imaging in living cells and tissues Xiuqi Kong, Baoli Dong, Nan Zhang, Chao Wang, Xuezhen Song, Weiying Lin www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(17)30655-0 http://dx.doi.org/10.1016/j.talanta.2017.06.023 TAL17642

To appear in: Talanta Received date: 13 March 2017 Revised date: 5 June 2017 Accepted date: 10 June 2017 Cite this article as: Xiuqi Kong, Baoli Dong, Nan Zhang, Chao Wang, Xuezhen Song and Weiying Lin, A unique red-emitting two-photon fluorescent probe with tumor-specificity for imaging in living cells and tissues, Talanta, http://dx.doi.org/10.1016/j.talanta.2017.06.023 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 galley proof before it is published in its final citable 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.

A unique red-emitting two-photon fluorescent probe with tumor-specificity for imaging in living cells and tissues Xiuqi Kong, Baoli Dong, Nan Zhang, Chao Wang, Xuezhen Song, Weiying Lin* Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, University of Jinan, Jinan, Shandong 250022, P. R. China

*

Corresponding author. Tel./fax: +86 531 82769108. [email protected]

1

ABSTRACT

Tumor-specific imaging can provide an attractive approach for the early detection and prognosis of cancer, as well as the precise image-guided tumor-removal surgery. Herein, we describe a unique red-emitting two-photon fluorescent probe (N-BN) for tumor-specific imaging. N-BN utilized Nile Red as the red-emitting two-photon fluorophore and employed biotin as the tumor-specific ligand. In the presence of a variety of biomolecules or in different pH buffers, the fluorescence intensity of N-BN at 655 nm showed no noticeable change. N-BN exhibited the remarkable two-photon absorption cross sections of 15.4 GM under excitation at 800 nm. Under the guidance of biotin, N-BN can be applied for the imaging of biotin-receptor positive cancer cells over biotin-negative cells under red-emitting one- and two-photon manners. Assisted by high-definition three-dimensional imaging, the living tumor tissues loaded with N-BN could display strong red two-photon fluorescence with a penetration depth of about 90 μm. Moreover, the in vivo and ex vivo imaging studies intuitively revealed that N-BN could track the tumor with highly tumor-specific property by a near-infrared manner. Graphical abstract

2

Abbreviations HOBT, 1-hydroxybenzotriazole; EDC, N-(3-Dimethylaminopropyl)-N'-ethylcarbod -iimide hydrochloride; DIEA, Ethyldiisopropylamine; DMF, Dimethylformamide. Keywords Red-emitting two-photon fluorescent probe; High-definition three-dimensional imaging; Tumor-specific imaging; in vivo fluorescence imaging

3

1. Introduction Cancer is one of the deadliest diseases to human all over the world, as there is usually no obvious symptoms until the diseases have got to an advanced stage [1]. It is convinced that early detection and prognosis of cancer could improve the quality of life of persons living with cancer [2-7].The traditional techniques for cancer detection are magnetic resonance imaging (MRI), computed tomography (CT) and positron emission tomography (PET) [8-9]. However, these modalities usually lack cancer-specificity, and make human suffered from danger of radiation. Thus, it is critical to explore more effective techniques to detect cancers. Fluorescence imaging is an emerging technique and has attracted intense interest for oncology imaging and diagnosis due to its high sensitivity, low-cost, low toxicity and the capability of imaging in real-time. To date, some fluorescent probes for cancer imaging have been developed [10-12]. However, these fluorescent probes are mostly operated under one-photon manner with short excitation, which is generally limited by high photo-damage and the shallow depth of tissue penetration. In contrast, the two-photon microscopy utilizes two NIR photons as the excitation source, which possesses many attractive advantages including minimum photodamage, deep penetration in tissues and high-definition three-dimensional imaging [13-15]. Currently, the common two-photon fluorophores (coumarin and naphthalimide, etc.) have been widely used in fluorescence imaging [16-19]. However, the emission wavelengths of these two-photon agents are mostly in blue (450-495 nm) or green region (495-570 nm), which are liable to be interfered with the auto-fluorescence in organisms and easily absorbed by biomolecules, such as hemoglobin and bilirubin [20-22]. To settle these matters, the red emission (600-750nm) is preferred because the long wavelength could decrease the interference from the autofluorescence and absorption by biomolecules [23-25]. Therefore, the development of the red-emissive two-photon fluorescent probe is highly important for cancer imaging. Nile Red (Nile) is a well-known two-photon fluorescent dye with red emission [26-28], which can provide favorable imaging with low background, high sensitivity 4

and deep penetration. Nile has been used in drug delivery system with red-emitting two-photon property [29]. Biotin, a necessary element to maintain the natural growth of cells, is an important member of tumor targeting group, as cancer cells over-express biotin receptors and require more biotin to sustain their rapid growing [30]. The biotin unit is an effective tumor-targeting ligand for cancer imaging, and it has been successfully applied for tracking cancer cells and designing targeted drug delivery [31]. To the best of our knowledge, the two-photon tumor-specific fluorescent probe with red emission for cancer imaging has not been reported. Herein, we describe the first example of red-emitting two-photon fluorescent probe (N-BN) with tumor-specificity for cancer imaging. In N-BN system, Nile was employed as the red two-photon fluorophore and biotin was selected as tumor-targeting group. N-BN displayed desirable stability in the presence of a variety of biomolecules or in different pH buffers. N-BN exhibited the remarkable two-photon absorption cross sections of 15.4 GM under excitation at 800 nm. Under the guiding of biotin, N-BN showed higher affinity to cancer cells relative to normal cells. High-definition 3D imaging in living tumor tissues was also successfully performed under the red two-photon mode. Besides, N-BN could detect tumor in a one-photon manner in vivo. The successful imaging of red two-photon fluorescent probe (N-BN) in vitro and in vivo provides an experimental evidence for cancer imaging. 2. Experimental 2.1. Materials and instruments All solvents and reagents used in synthesis were obtained from commercial suppliers without further purification. Dulbecco's Modified Eagle Media (DMEM) and fetal bovine serum (FBS) were from Gibico, Nile and biotin were from Meilunbio. All compounds were monitored and purified by thinner layer chromatography (TLC) and silica gel (200-300 mesh) column, then characterized by NMR and High Resolution Mass Spectrum (HRMS). 1HNMR and 13CNMR were performed with the 5

Bruker spectrometer (400 MHz), and HRMS was obtained with Bruker 9.4T Apex-ultra hybrid Qh-FTICR. The absorption spectra were carried out with the Shimadzu UV-2700 UV-Vis Spectrophotometer, and fluorescent spectra were collected by the Hitachi F4600 Fluorescent Spectrophotometer. Cell cytotoxicity was carried out with Thermo Scientific Multiskan FC Microplate reader. Imaging in vitro experiments were performed with Nikon A1 Confocal Laser Scanning Microscope (CLSM) equipped with a femtosecond laser for two-photon imaging. In vivo and ex vivo experiments were received with Caliper Life Sciences animal imaging system.

2.2. Synthesis of N-BN Compound 2 (N-OH) was synthesized according to previously reported methods [32].Compound 2 (334 mg, 1 mmol), biotin (244 mg, 1 mmol), HOBT (337.5 mg, 2.5 mmol), EDC (270 mg, 1.5 mmol) and DIEA (0.3 mL) were mixed in DMF (5.0 mL) and stirred overnight. Then 10 mL water was added into the mixture and extracted with CH2Cl2, washed with water, dried over Na2SO4 and evaporated under reduced pressure. Purification by column chromatography (CH2Cl2: MeOH = 15:1) to afford N-BN (190 mg) as a dark red solid. Yield: 35%.1H NMR (CDCl3, 400 MHz): δ 8.31 (m, 2 H), 7.54 (d, J= 8.8 Hz, 1 H), 7.35 (dd, 1 H), 6.61 (m, 1 H), 6.42 (d, J = 2.4 Hz, 1 H), 6.34 (s, 1 H), 6.03 (s, 1 H), 5.42 (s, 1 H), 4.55 (t, J = 7.2 Hz, 1 H), 4.38 (t, J = 4.8 Hz, 1 H), 3.46 (q, 4 H), 3.22 (q, 1 H), 2.96 (m, 1 H), 2.80 (d, J = 8.8 Hz, 1 H), 2.68 (t, J = 7.6 Hz, 2 H), 1.86 (m, 3 H), 1.59 (m, 3 H), 1.26 (t, J = 7.2 Hz, 6 H).

13

C

NMR (CDCl3, 100 MHz): 182.7, 171.9, 163.8, 153.1, 152.2, 150.9, 146.8, 138.8, 133.6, 131.2, 129.3, 127.5, 124.8, 123.4, 116.3, 109.9, 105.3, 96.1, 62.0, 60.1, 55.5, 45.1, 40.6, 34.0, 28.4, 28.3, 24.7, 12.6. HRMS (ESI): m/z calculated for C 30H32N4O5S 561.2166 [M + H] +, found: 561.2200; 583.1991 [M + Na] +, found: 583.1989. 2.3. Preparation of testing solutions The stock solutions of Nile, N-OH and N-BN were 1 mM in DMSO. 5 μM of testing solutions of three probes were prepared with Britton-Robinson (B-R) buffer 6

(10% DMSO, pH=7.4) respectively. Solutions of testing in different pH (4.5-10.0) were prepared by B-R buffer according to standard methods. Solutions with various interference species were prepared with 100 μM of Ca2+, Co2+, Cu2+, Fe2+, Fe3+, Zn2+, Mg2+,Ni2+, Na+,NO2-, NO3-, GSH, H2O2,HCys, NO, Vc, Na2S, Na2SO3 and Glucose respectively. 2.4. Two-photon absorption cross section assay The relative two photon absorption cross section were determined according to the previously reported methods [33]. 5 μM Nile, N-OH,N-BN and Rhodamine 6G (a standard) were prepared in B-R buffer (10% DMSO, pH=7.4)，then they were measured with femto-second laser. The relative two-photon absorption cross section (δ) was calculated by this equation: δs = δr(SsΦrϕrcr)/(SrΦsϕscs), where the subscripts s and r stand for the sample and standard (Rhodamine 6G).S was the intensity of the signal collected using a CCD detector. Φ was the fluorescence quantum yield, and ϕ was the fluorescence collection efficiency of the experimental apparatus. c was the concentration. δr was the two-photon absorption cross section of Rhodamine 6G. 2.5. Cell culture and cytotoxicity assay HeLa, 4T-1, A549 and NIH 3T3 cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotics (100 U/mL penicillin and 100μg/mL streptomycin) at 37 °C and 5% CO2. The MTT assay was used to evaluate the cytotoxicity of N-OH and N-BN. The above cells were seeded into 96-well plates at a density of 1×104 cells/well and incubated for 18 h. The media were replaced by fresh culture contained both sensors over a range of concentrations (0 - 30 μM). After18h incubation, 10μL of MTT (5 mg/mL in PBS) was added and then incubated another 4 h. After that, the media was removed, and 100μL of DMSO was added to dissolve the formazan crystals. The plate was shaken for 10 min, and each well was finally analyzed by the microplate reader and detected at the absorbance of 490 nm. 2.6. Red-emitting one and two-photon cellular imaging study 7

Before experiments, biotin receptor over-expressed positive cells (HeLa, 4T-1 and A549) and biotin receptor low-expressed negative cells (NIH 3T3) were placed into 35 mm glass bottom dishes for 24 h at the confluence of 50%. The stock solutions of N-OH and N-BN were 1 mM in DMSO. 5 μM of N-OH or N-BN culture was prepared with DMEM supplemented with 10% FBS (5% DMSO). Then, 1 mL of 5 μM N-BN or N-OH fresh culture were added into dishes to replace the previous culture respectively for incubation another 15 min. After being washed with PBS twice, cell imaging was taken by CLSM. For competition experiments, positive cells were pre-treated with biotin (1 mM) for 30 min，then N-BN (5 μM) was added into cells for further incubation 15 min for detecting the imaging. For one-photon imaging, fluorescence was acquired with excitation at 561 nm and emission at the range of 570-620 nm. For red two-photon imaging, fluorescence was acquired with excitation at 800 nm and emission at the range of 570-620 nm. 2.7. Xenograft tumor model preparation and red-emitting two-photon imaging in living tumor tissues All animal studies were approved by the Institutional Animal Care and Use Committee of the Shandong University. Four-week old female Balb/c mice were purchased from School of Pharmaceutical Sciences, Shandong University. They were kept in a temperature-controlled environment with 12 h light/dark cycle. For subcutaneous xenograft establishment, fur were removed by an electric shaver at first, then the 5 x 105 of 4T-1 cells were injected at right flank in Balb/c mice. Mice were kept about 10 to 15 days before experiments when tumors were up to about 0.5 cm in diameter. For living tumor tissues imaging study, tumors were excised from tumor bearing mice and cut into small slices. The stock solutions of N-OH and N-BN were 2 mM in DMSO. 10 μM of N-OH or N-BN media were prepared with PBS (5% DMSO). Then slices were incubated with N-BN or N-OH (10 μM) for 1 h. Then slices were incubated with N-BN or N-OH (10 μM) for 1 h. After washed with PBS three times, the slices were performed NIR and two-photon imaging by CLSM 8

equipped with femto-second laser. For two-photon imaging, fluorescence was acquired with excitation at 800 nm and emission at the range of 570-620 nm. 2.8. NIR imaging in vivo and ex vivo For in vivo imaging, mice received N-BN or N-OH at a dose of 1mg/kg body weight. The stock solutions of N-OH and N-BN were 10 mM in DMSO. 100 μL of N-OH or N-BN media were prepared with PBS (5% DMSO), then injected into mice by intratumor injection for the imaging. Before imaging, mice were anesthetized with isoflurane. Then the fluorescence was collected with IVIS Lumina XR system. After about 6 h, imaging was performed using a 600 nm excitation and a 670 nm emission filter. The fluorescence imaging settings (exposure time, 1 seconds; Binning, medium; F-stop, 2). For ex vivo imaging, after achieving in vivo experiments, mice were euthanized by cervical dislocation methods, tumors and other organs (heart, lung, liver, spleen, kidney) were excised and imaged under IVIS Lumina XR system using a 600 nm excitation and a 670 nm emission filter.

3. Results and discussion 3.1. Design and synthesis of N-BN and N-OH To design a novel red two-photon fluorescence probe, Nile was selected as a red two-photon platform. Biotin was employed as a targeting ligand for guiding the probe to accumulate around tumors. However, Nile is difficult to conjugate with other groups, so we constructed the N-OH according to previously reported synthetic methods [32]. With these considerations, N-OHwas conjugated with biotin through esterification reaction to form an ideal red two-photon probe N-BN (Scheme 1a). As displayed in Scheme 1b, N-BN could enter cancer cells by biotin receptor mediated endocytosis, and image cells and tissues under red two-photon manner.

3.2. Spectroscopic properties of N-BN

9

Initially, the optical properties of Nile, N-OH and N-BN were investigated using UV-Vis absorption and fluorescence spectra. As shown in Fig.1a, Nile showed a maximum absorption at 595 nm with molar extinction coefficient (ε) of 0.37×104 M-1 cm-1 in B-R buffer (10% DMSO, pH 7.4), and N-OH displayed a maximum absorption at 595 nm with ε of 0.65×104 M-1 cm-1. Compared with Nile and N-OH, N-BN showed a blue-shifted absorption at 545 nm (ε= 1.8×104 M-1 cm-1), which can be ascribed to the π-π* transition of the benzophenoxazine group. Under excitation at 595 or 545 nm, N-BN, N-OH and Nile all showed strong fluorescence at about 655 nm which was in red region, and possessed the fluorescence quantum yields of 0.21, 0.64 and 0.42, respectively (Fig. 1b and Fig.S1). It indicates that the introduction of biotin group exerts no significant influence to the fluorescence of N-OH. Therefore, N-BN showed desirable fluorescence properties and can be potentially applied for imaging.

Next, the stability of the fluorescence spectra of N-BN and N-OH in presence of various relevant biomolecules or in different pH conditions was explored. As shown in Fig. 2a and Fig. S2, N-BN and N-OH displayed similar fluorescence spectra in pH 4.5-10. Meanwhile, the fluorescence of N-BN and N-OH had nearly no noticeable change after the treatment with a variety of molecules including Ca2+, GSH, Cys and Vc (Fig. 2b and Fig.S3). These data suggest that the pH and relevant biomolecules showed no marked disturbance to the fluorescence properties of N-BN. Thus, N-BN and N-OH maybe used for imaging in the physiological conditions.

Notably, for a two-photon bioimaging probe, the two-photon absorption cross section (δΦ) is a key parameter. In these experiments, the δΦ values for N-BN, N-OH and Nile were determined at the emissive wavelengths ranging from 570-620 nm in B-R buffers (10% DMSO, pH 7.4). As depicted in Fig. 3, N-BN, N-OH and Nile showed the δΦ values of 15.4, 16.8 and 24.4 under excitation at 800 nm. Thus, N-BN 10

can be served as a two-photon agent with red emission for living cell and tissue imaging.

3.3. Cell cytotoxicity study Encouraged by spectroscopic results of N-BN and N-OH, we investigated the cytotoxicity of N-BN and N-OH to HeLa, A549, 4T-1 and NIH 3T3 cells with MTT methods. As illustrated in Fig.S4, both probes with various concentrations (1-30 μM) were almost no cytotoxicity to these four kinds of cells after 18 h incubation. The cell viability rates were still over 90% even at 30 μM. Cytotoxicity assay indicates N-BN and N-OH have good biocompatibility and are suitable for imaging in living cells.

3.4. Red-emitting one-and two-photon cellular imaging study Inspired by the spectra data and cellular cytotoxicity assays, we then performed the imaging experiments with N-BN and N-OH in living cells. HeLa, 4T-1 and A549 were incubated with 5 μM of N-BN and N-OH for 15 min to test the potential specificity. As expected, HeLa, 4T-1 and A549 cells loaded with N-BN displayed stronger fluorescence than these cells treated with N-OH (Fig.4 and Fig. S5) under the one-photon mode. The relative quantified fluorescence intensities demonstrated that N-BN groups were about 1-1.4 fold greater than the N-OH groups (Figs.4b, d and Figs.S5c, d), confirming that biotins can facilitate endocytosis of N-BN in positive cancer cells. Meanwhile, NIH 3T3 cells with low-expressed biotin receptors, showed weaker fluorescence after the incubation of N-BN (Fig. 4andFig. S5). Furthermore, biotin competition experiments were also carried out to verify the tumor-specificity of N-BN. HeLa, 4T-1 and A549 cells were pre-incubated with excess biotin to make receptor saturation, and then treated with N-BN for another 15 min. As shown in Fig.4 and Fig. S5, these cells displayed dramatically reduced fluorescence of N-BN, which further reveals that the biotin unit plays an essential role in the endocytosis of N-BN. Thus, N-BN can serve as an effective tumor-specific agent for the of cancer cell imaging. 11

Importantly, the two-photon absorption cross section experiments showed N-BN was of desirable red-emitting two-photon feature, so the red-emitting two-photon fluorescence imaging of N-BN was also performed in above cells simultaneously. As illustrated in Fig. 5 and Fig. S6, HeLa, 4T-1, A549 cells loaded with N-BN gave stronger fluorescence with red-emission than these cells incubated with N-OH in two-photon channel (λex = 800 nm, λem = 570-620 nm), and the quantified results were further confirmed this phenomenon (Figs. 5b, d and Fig. S6b). These data were perfectly in accordance with the above-mentioned one-photon results. Taken together, under the guiding of biotin, N-BN was capable of tracing cancer cells in red-emitting two-photon channel and one-photon channel.

3.5. Red-emitting two-photon imaging in tumor tissues study In view of the exciting imaging results in living cancer cells, we next extended applications of N-BN and N-OH in living tumor tissues in red-emitting two-photon channel. As shown in Fig. 6a, after the treatment with10 μM N-BN for 1h, the tumor tissues showed strong red two-photon fluorescence with the penetration depth of about 90 μm. The three-dimensional reconstruction imaging in Fig. 6b vividly reflected individual layer image. Under the same conditions, the tumor tissues treated with N-OH, exhibited weak fluorescence with the penetration depth of 60 μm (Figs. 6c and 6d). Moreover, the depth of tissues treated with N-BN was deeper than N-OH treated tissues. Therefore, N-BN displayed the ability of specificity in imaging tumor tissues under the red two-photon manner.

3.6. NIR imaging study in vivo and ex vivo With the imaging results obtained from cells and tissues in hand, we sought to study imaging in vivo with N-BN and N-OH. The tumor-bearing mice were injected with N-BN or N-OH, and fluorescence was detected with an in vivo imaging system at an excitation wavelength of 600 nm and emission wavelength of 670 nm. As 12

displayed in Fig. 7a, mice loaded with N-BN displayed marked fluorescence around the tumor after 6 h, while the mice injected with N-OH gave weak fluorescence in NIR manner. This probably because biotin can facilitate N-BN to accumulate around the tumor. The in vivo and ex vivo results were consistent with the imaging of cells and tissues, suggesting N-BN is capable of in vivo imaging tumor with NIR manner.

Next, we studied the sub-distribution of N-BN and N-OH in organs. The mice were euthanized after imaging in vivo, then organs (heart, kidney, lung, liver and spleen) and tumors were excised, and imaged at an excitation wavelength of 600 nm and emission wavelength of 670 nm. As depicted in the Fig. 7b, the remarkable fluorescence was observed in tumors and liver, and almost no fluorescence was observed in heart, kidney, lung and spleen. This demonstrates that N-BN can trace the tumor under NIR manner. Meanwhile, the tumor loaded with N-BN showed stronger fluorescence than the tumor treated with N-OH. This implies the biotin can facilitate N-BN to accumulate around tumors. Therefore, N-BN can be used for in vivo tumor imaging.

13

4. Conclusion In summary, we have developed a unique red two-photon probe N-BN with tumor-specificity. N-BN showed well stability in the presence of various biomolecules or in different pH buffer. The two photon absorption cross section experiments of N-BN displayed its satisfactorily red-emitting two-photon feature. Assisted by biotin, N-BN can be selectively applied for imaging in biotin-receptor positive cancer cells over biotin-negative cells under red-emitting one- and two-photon manners. N-BN showed favorable tumor-selectivity in cancer cells imaging. Notably, the living tumor tissues loaded with N-BN displayed strong red two-photon fluorescence with a penetration depth of about 90 μm. Moreover, the in vivo and ex vivo imaging studies intuitively revealed that N-BN can track the tumor with highly tumor-specific property under a near-infrared manner.

Acknowledgments This work was financially supported by NSFC (21472067, 21672083, 51602127), Taishan Scholar Foundation (TS 201511041), the startup fund of the University of Jinan (309-10004), the Doctoral Fund of University of Jinan (160100135), and Science and Technology Program of University of Jinan (140200125).

14

References [1] S. S. Buys, E. Partridge, A. Black, C. C. Johnson, L. Lamerato, C. Isaacs, D. J. Reding, R. T. Greenlee, L. A. Yokochi, B. Kessel, E. D. Crawford, T. R. Church, G. L. Andriole, J. L. Weissfeld, M. N. Fouad, D. Chia, B. O'Brien, L. R. Ragard, J. D. Clapp, J. M. Rathmell, T. L. Riley, P. Hartge, P. F. Pinsky, C. S. Zhu, G. Izmirlian, B. S. Kramer, A. B. Miller, J. L. Xu, P. C. Prorok, J. K. Gohagan, C. D. Berg, Effect of screening on ovarian cancer mortality: the prostate, lung, colorectal and ovarian (PLCO) cancer screening randomized controlled trial, JAMA- J. Am. Med. Assoc. 305(2011) 2295-303.. [2] L. Fass, Imaging and cancer: a review, Mol. Oncol. 2(2008) 115-152. [3] R.A. Smith, A. C. von Eschenbach, R. Wender, American Cancer Society guidelines for the early detection of cancer: update of early detection guidelines for prostate, colorectal, and endometrial cancers: Also: update 2001-testing for early lung cancer detection, CaA Cancer J. Clin. 51(2001) 38-75. [4] W. Willett, Cancer prevention and early detection, Cancer Epidemio. Biomar. 12 (2003) 69-70. [5] C. Mettlin, F. Lee, J. Drago, G. P. Murphy, The American Cancer Society national prostate cancer detection project. Findings on the detection of early prostate cancer in 2425 men, Cancer 6(1991) 2949-2958. [6] K. S. Anderson, S. Sibani, G. Wallstrom, J. Qiu, E.A. Mendoza, J. Raphael, E. Hainsworth, W. R. Montor, J. Wong, J. G. Park, N. Lokko, T. Logvinenko, N. Ramachandran, A. K. Godwin, J. Marks, P. Engstrom, J. Labaer, Protein microarray signature of autoantibody biomarkers for the early detection of breast cancer, J. Proteome Res. 10(2010) 85-96. [7] R. A. Smith, V. Cokkinides, A. C. von Eschenbach, et al. American Cancer Society guidelines for the early detection of cancer, CaA Cancer J. Clin. 54 (2002) 11-25. [8] R. Weissleder, M. J. Pittet, Imaging in the era of molecular oncology, Nature 452 (2008) 580-589. 15

[9] T. H. Oude Munnink, W. B. Nagengast, A. H. Brouwers, C. P. Schröder, G. A. Hospers, et al. Molecular imaging of breast cancer. Breast 18(2009)66-73. [10] G. M. van Dam, G. Themelis, L. M. Crane, N. J. Harlaar, R. G. Pleijhuis, W.

Kelder, A. Sarantopoulos, J. S. de Jong, H. J. Arts, A. G. van der Zee, J. Bart, P. S. Low, V. Ntziachristos, Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results, Nat. Med. 17(2011) 1315-1319. [11] E. Bellard, S. Chabot, V. Ecochard, O. Martinez, S. Pelofy, L. Paquereau, M. Rols,

B. Couderc, J. Teissie, M. Golzio, Fluorescence imaging in cancerology, Radiol. Oncol. 2(2013) 3-17. [12] B. Ballou, L.A. Ernst, A.S. Waggoner, Fluorescence imaging of tumors in vivo, Curr. Med. Chem. 12(2005) 795-805. [13] W. Denk, J. H. Strickler, W. W. Webb, Two-photon laser scanning fluorescence microscopy, Science 248 (1990)73-76 [14] F. Helmchen, W. Denk, Deep tissue two-photon microscopy, Nat. Methods 2(2005) 932-940. [15] H. M. Kim, B. R. Cho, Small-molecule two-photon probes for bioimaging applications, Chem. Rev. 115(2015) 5014-5055. [16] B. Dong, X. Song, X. Kong, C. Wang, Y. Tang, Y. Liu, W. Lin, Simultaneous near-infrared and two-photon in vivo imaging of H2O2 using a ratiometric fluorescent probe based on the unique oxidative rearrangement of oxonium, Adv. Mater. 28(2016) 8755-8759. [17] X. H. Zhou, Y. R. Jiang, X. J. Zhao, D. Guo, A naphthalene-based two-photon fluorescent probe for selective and sensitive detection of endogenous hypochlorous acid, Talanta 160 (2016) 470-474. [18] Y. Tang, X. Kong, A. Xu, B. Dong, W. Lin, Development of a Two‐Photon Fluorescent Probe for Imaging of Endogenous Formaldehyde in Living Tissues, Angew. Chem. Int. Edit., 128(2016) 3356-3359.

16

[19] J. F. Zhang, C. S. Lim, S. Bhuniya, B. R. Cho, J.S. Kim, A highly selective colorimetric and ratiometric two-photon fluorescent probe for fluoride ion detection, Org. Lett. 13(2011)1190-1193. [20] J. Hung, S. Lam, J. C. Leriche, B. Palcic, Autofluorescence of normal and malignant bronchial tissue, Lasers Surg. Med. 11(1991) 99-105. [21] P. Datta, Effect of Hemolysis, High Bilirubin, Lipemia, Paraproteins, and System Factors on Therapeutic Drug Monitoring. Totowa, NJ. 2008. [22] L. A. Pyles, E. J. Stejskal, S. Einzig, Spectrophotometric measurement of plasma 2-thiobarbituric acid-reactive substances in the presence of hemoglobin and bilirubin interference, Exp. Biol. Med. 202(1993) 407-419. [23] H. Kobayashi, M. Ogawa, R. Alford, P. L. Choyke, Y. Urano, New strategies for fluorescent probe design in medical diagnostic imaging, Chem. Rev. 110(2009) 2620-2640. [24] H. Chen, B. Dong, Y. Tang, W. Lin, A Unique “Integration” Strategy for the Rational Design of Optically Tunable Near-Infrared Fluorophores, Accounts Chem. Res. (2017) DOI: 10.1021/acs.accounts.7b00087. [25] H. Li, L. Guan, X. Zhang, H. Yu, D. Huang, M. Sun, S. Wang, A cyanine-based near-infrared fluorescent probe for highly sensitive and selective detection of hypochlorous acid and bioimaging, Talanta 161(2016) 592-598. [26] M. B. Brown, T. E. Edmonds, J. N. Miller, N. J. Seare, Use of Nile Red as a long-wavelength fluorophore in dual-probe studies of ligand-protein interactions, J. Fluoresce. 3(1993)129-130. [27] C. Sissa, V. Parthasarathy, D. Drouin-Kucma, M. H. Werts, M. Blancharddesce, F. Terenziani, The effectiveness of essential-state models in the description of optical properties of branched push-pull chromophores, Phys. Chem. Chem. Phys.12(2010) 11715-11727. [28] S. Satapathi, H.S. Gill, L. Li, L. Samuelson, J. Kumar, Two-Photon Active Nile Red Loaded Fluorescent Polystyrene Nanoparticles, Adv. Sci. Focus 1(2013) 3-5. 17

[29] R. F. Donnelly, D. I. J. Morrow, F. Fay, C. J. Scott, S. Abdelghany, R. R. Singh, M. J. Garland, A. D. Woolfson, Microneedle-mediated intradermal nanoparticle delivery: potential for enhanced local administration of hydrophobic pre-formed photosensitisers, Photodiagnosis Photodyn. Ther. 7(2010) 222-231. [30] P. M. West, W. H. Woglom, Abnormalities in the distribution of biotin in certain tumors and embryo tissues, Cancer Res. 2(1942) 324-331. [31] M. Li, J. W. Y. Lam, F. Mahtab, S. Chen, W. Zhang, Y. Hong, J. Xiong, Q. Zheng, B. Tang, Biotin-decorated fluorescent silica nanoparticles with aggregation-induced emission characteristics: fabrication, cytotoxicity and biological applications, J. Mater. Chem. B 1(2013) 676-684. [32] J. Jose, K. Burgess, Syntheses and properties of water-soluble Nile Red derivatives, J. Org. Chem. 71(2006) 7835-7839. [33] N. S. Makarov, M. Drobizhev, A. Rebane, Two-photon absorption standards in the 550 -1600 nm excitation wavelength range, Opt. Express 16(2008) 4029-4047.

18

Scheme 1. Design strategy of the red-emitting two-photon probe for tumor-specific imaging. (a) The molecular structure of Nile and its derivatives (N-OH and N-BN). (b) Schematic illustrates the uptake of N-BN in cancer cells.

Fig. 1. The UV-Vis absorption and fluorescence spectra of N-BN, N-OH and Nile. (a) The UV-Vis absorption spectra of 5 μM N-BN, N-OH and Nile in B-R Buffer (10% DMSO). (b) Fluorescence spectra of 5 μM N-BN, N-OH and Nile in B-R Buffer (10% DMSO) excited at 590 nm.

Fig. 2. (a) Fluorescence intensities of N-BN (5 μM) at 655 nm in various pH buffer excited at 590 nm. (b) Fluorescence intensities of N-BN (5 μM) at 655 nm containing different species in the B-R buffer (10% DMSO, pH7.4) excited at 590 nm. (1) blank, (2) Ca2+, (3) Co2+, (4) Cu2+, (5) Cys, (6) Fe2+, (7) Fe3+, (8) GSH, (9) Hcys, (10) NO3-, (11) Mg2+, (12) Na+, (13) S2-, (14) SO32-, (15) NO2-,(16) NO,(17) VC,(18) Zn2+, (19) Ni2+, (20) Glucose. All species were 100 μM. Error bars represent standard deviation (±S.D.), n = 3.

Fig. 3. Two photon absorption cross section (δΦ) spectra of N-BN, N-OH and Nile in B-R buffers (10% DMSO, pH 7.4).

Fig. 4. Tumor-specific imaging in HeLa and NIH 3T3cells under one-photon mode. (a) Fluorescence images of HeLa cells treated with 5 μM of N-BN, 1mM of biotin and N-BN, N-OH respectively. (b) Fluorescence images of NIH 3T3 cells treated with 5 μM of N-BN and N-OH respectively. (c) Quantified fluorescence intensities of (a) relative to N-OH treated groups in the TRITC channel. (d) Quantified fluorescence intensities of (c) relative to N-OH treated groups in the TRITC channel. TRITC: λex = 561 nm, λem = 570-620 nm. Scale bar: 20 µm. Error bars represent standard deviation (±S.D.), n = 3. Statistical analyses were analyzed by one-way ANOVA method, the 19

significance was assumed at p value less than 0.05.

Fig. 5. Red-emitting two-photon imaging in HeLa and NIH 3T3 cells. (a) Imaging of HeLa cells treated with 5 μM of N-BN, 1mM of biotin and N-BN, N-OH respectively. (b) Quantified fluorescence intensities of (a) relative to N-OH treated groupsin the TRITC channel. (c) Imaging of NIH 3T3 cells treated with 5 μM of N-BN and N-OH respectively. (d) Quantified fluorescence intensities of (c) relative to N-OH treated groups in the TRITC channel. TRITC: λex = 800 nm, λem = 570 - 620 nm. Scale bar: 20 µm. Error bars represent standard deviation (±S.D.), n = 3. Statistical analyses were analyzed by one-way ANOVA method, the significance was assumed at p value less than 0.05.

Fig. 6. Red-emitting two-photon imaging of living tumor tissue stained with 10 μM N-BN and N-OH for 1 h. (a) Representative image of N-BN group at a depth of 40 μm, λex = 800 nm, λem = 570 - 620 nm, scale bar: 50 μm. (b) 3D imaging of all images at two-photon mode of N-BN group. (c) Representative image of N-OH group at a depth of 40 μm, λex = 800 nm, λem = 570 - 620 nm, scale bar: 50 μm. (d) 3D imaging of all images at two-photon mode of N-OH group.

Fig. 7. (a) In vivo imaging of tumor-bearing mice at 6 h after injection of N-BN and N-OH (1mg / kg). (b) Sub-distribution imaging of the internal organs after anatomy for N-BN group and N-OH group. λex = 600 nm, λem = 670 nm.

20

Scheme 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Highlights



We have constructed a novel red-emitting two-photon and tumor-specific fluorescent probe N-BN for tumor imaging.



The probe N-BN exhibits remarkably tumor-specificity under one-and two-photon modes with red emission in overexpressed biotin-receptors cancer cells over negative cells.



The probe N-BN demonstrates satisfactorily red-emitting two-photon property by achieving high-definition 3D imaging with the penetration depth of about 90 μm in living tumor tissues.

21