A Two-Photon Ratiometric Fluorescent Probe for Imaging

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A two-photon ratiometric fluorescent probe for imaging carboxylesterase 2 in living cells and tissues Qiang Jin, Lei Feng, Dan-Dan Wang, Zi-Ru Dai, Ping Wang, Li-Wei Zou, Zhihong Liu, Jia-yue Wang, Yang Yu, Guang-Bo Ge, Jing-Nan Cui, and Ling Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09573 • Publication Date (Web): 07 Dec 2015 Downloaded from http://pubs.acs.org on December 14, 2015

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ACS Applied Materials & Interfaces

A two-photon ratiometric fluorescent probe for imaging carboxylesterase 2 in living cells and tissues Qiang Jin,a,d Lei Feng,b,d Dan-Dan Wang,a Zi-Ru Dai,a Ping Wang,a Li-Wei Zou,a Zhihong Liu,c Jia-yue Wang,a Yang Yu,a Guang-Bo Ge,*a Jing-Nan Cui*b and Ling Yang*a a

Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.

b

State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian, China.

c

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, China. ABSTRACT: In this study, a two-photon ratiometric fluorescent probe NCEN has been designed and developed for highly selective and sensitive sensing of human carboxylesterase 2 (hCE2), on the basis of the catalytic properties and substrate preference of hCE2. Upon addition of hCE2, the probe could be readily hydrolyzed to release 4-amino-1, 8-naphthalimide (NAH) which brings remarkable red-shift in fluorescence (90 nm) spectrum. The newly developed probe exhibits good specificity, ultrahigh sensitivity and has been successfully applied to determine the real activities of hCE2 in complex biological samples, such as cell and tissue preparations. NCEN has also been used for two-photon imaging of intracellular hCE2 in living cells as well as in deep-tissues for the first time, and the results showed that the probe exhibited high ratiometric imaging resolution and deep-tissue imaging depth. All these findings suggested that this probe holds great promise for applications in bio-imaging of endogenous hCE2 in living cells and in exploring the biological functions of hCE2 in complex biological systems. KEYWORDS: two-photon, ratiometric fluorescent probe, carboxylesterase 2, specific sensing, bio-imaging INTRODUCTION Carboxylesterases (CEs) are members of α/β hydrolase fold proteins, which are mainly localized in the endoplasmic reticulum of various mammalian cells.1-3 CEs participate in hydrolysis of numerous xenobiotics and endogenous ester- or amide-containing compounds.4-6 In human, two major CE isoforms have been identified as human carboxyleserase 1 (hCE1) and human carboxyleserase 2 (hCE2).7, 8 These two isozymes display notably difference on the basis of tissue distribution and substrate specificity. Generally, hCE1 is mainly expressed in liver, and prefer to hydrolyze substrates with small alcohol groups and large acyl groups.9, 10 In contrast to hCE1, hCE2 is mostly expressed in small intestine and mainly recognizes esters with large alcohol and small acyl group.11-13 As the major CE isoform distributed in human intestine and tumour tissues, hCE2 plays key roles in oral bioavailability of ester drugs and the treatment outcomes of several anticancer prodrugs (such as irinotecan and capecitabine).14-16 Notably, hCE2 is highly expressed in many human tumour cells, the expression and function of hCE2 in tumour cells is essential to the activation and susceptibility of many anti-cancer prodrugs.17-19 Thus, the accurate measurement of the real activities of hCE2 in different biological systems (such as tissues and cancer cell lines) is of great importance for drug discovery and clinical practice.

In recent years, small molecule fluorescent probes for selective monitoring of a given target in biological samples are becoming increasingly attractive, due to their inherent advantages, such as high specificity, ultrasensitivity, non-destructiveness, easy management, as well as capability of being applicable to high-throughput screening.20-22 To date, several fluorescent probes for hCE2 have been reported and widely used in various in vitro assays.2326 However, all reported fluorescent sensors for hCE2 work with one photo microscopy (OPM), which can be interfered by absorption and auto-fluorescence of biological matrix, as well as other environmental factors. Furthermore, the single-photon excited fluorescent probes are hardly used in deep-tissue imaging because of the shallow penetration depth. Therefore, it is challenging and highly desired to develop novel chemical tools for deep tissue imaging and exploring the functions of hCE2 at subcellular level. To detect hCE2 in deep inside live tissues, it is crucial to use two-photon microscopy (TPM), a new technique that utilizes two photons of lower energy as the excitation source.27-29 Such method takes deeper penetration of excitation light beam into the complex biological tissues and provides better spatiotemporal resolution.30-32 In contrast to traditional single-photon excited fluorescent probes, two-photon excited (TPE) fluorescent probes are induced

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by two-photon excitation (TPE) with near-infrared (NIR) photons, which can facilitate 3D imaging, minimize autofluorescence background, reduce phototoxicity or photodamage to biological samples and increase specimen penetration.33 These advantages make TPM as a promising tool to investigate biomolecules in complex biological systems including living cells and turbid tissues. Unfortunately, to the best of our knowledge, TPE fluorescent probes for hCE2 have not been reported yet. Thus, it is necessary to develop a TPE probe for sensing hCE2 in living cells and deep tissues. In the present study, we report a ratiometric twophoton fluorescent probe (NCEN) derived from 4-amino-1, 8-naphthalimide (NAH) for imaging and sensing of hCE2 in live cells and tissues with high selectivity and sensitivity. This new probe not only can be readily prepared, but also shows excellent sensing properties. First, it is highly selective and sensitive for hCE2 over various analytes, including many hydrolases and common biological metallic ions or amino acids in human tissues or fluids. Second, it displays a rapid, colorimetric and ratiometric fluorescent detection process for hCE2 in aqueous solution under physical conditions. Last, this probe owns great advantages for bio-imaging endogenous hCE2 for its ability to avoid the auto-fluorescence of organisms and detect deeply into tissues. EXPERIMENTAL SECTION

1. Materials and chemicals. All chemicals were commercial products of analytical grade. Paraoxonases including PON1 and PON2 were obtained from bioworld, a1Acid Glycoprotein (AAG), dipeptidyl peptidase subtype including DPP-IV, DPP-VIII and DPP-IX, а-chymotrypsin (а-CT), carbomic Anhydrase I (CA), pepsin, trypsin, acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and human serum albumin (HSA) were all obtained from Sigma, lysozyme and fibroblast activation protein (FAP) were got from Solarbio and R&D System respectively. The recombinant CE isoforms including CE1b and CE1c were purchased from BD company. hCE2, Pooled human liver microsomes (HLM) and intestine microsomes (HIM) were received from RILD (Shanghai, China). Bis-p-nitrophenyl phosphate (BNPP), loperamide (LPA), fluorescein diacetate (FD) and fluorescein (F) were purchased from TCI (Tokyo, Japan). Ethylene diamine tetraacetic acid (EDTA) and huperzine A (HA) were obtained from J&K Chemical Ltd. (Beijing, China). HepG2 cell lines were provided by Cell bank (Shanghai, China). Stock solution of NCEN (20 mM) was prepared in DMSO and stored at -80 °C until use. Stock solutions (5 mg/mL) of enzymes were prepared in phosphate buffer (pH=7.4, 100 mM). NMR spectra were measured with a 400 MHz Bruker spectrometer set TMS as reference for 1H and 13C NMR. Accurate mass correction is measured with LTQ Orbitrap XL. Fluorescence emission/excitation spectra were measured on Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek). The hydrolysis supernatants were determined by a Shimadzu

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UFLC system coupled with a diode array detector and a mass spectrometer (Shimadzu 2010 EV, Japan). 2. Synthesis and structural characterization of NCEN. To a solution of 0.5 mmol 4-amino-N-n-butyl-1,8naphthalimide and 4-Dimethylaminopyridine (DMAP) (0.625 mmol) in 30 mL of CH2Cl2, 2-chloroacetyl chloride (0.75 mmol, mixed with 5 mL of CH2Cl2) was added dropwise at 0 °C in 30 min (Scheme S1). After stirring at this temperature for 1 h, the mixture was warmed to room temperature and stirred overnight. The solvent was removed in vacuo, and the residual solid was purified by chromatography (silica gel, dichloromethane as eluent) to afford 115.5 mg (67%) of NCEN as a pale-yellow solid. The structure of NCEN was confirmed by 1H-NMR, 13C-NMR and HRMS (Fig S1-3), and the data are as follows: 1H-NMR (CDCl3, 400 MHz) , 0.98 (t, J = 7.2 Hz, 3H), 1.39-1.48 (m, J = 7.2 Hz, 2H), 1.57-1.74 (m, J = 7.2 Hz, 2H), 4.16 (t, J = 7.2 Hz, 2H), 4.39 (s, 2H), 7.80 (t, J = 8.4 Hz, 1H), 8.16 (d, J = 8.5 Hz, 1H), 8.45 (d, J = 8.0 Hz, 1H), 8.61 (m, 2H), 9.15 (s, 1H, N-H).13C (CDCl3, 100 MHz) 164.16, 163.98, 163.48, 137.02, 132.11, 131.36, 128.88, 127.26, 125.66, 123.88, 123.64, 119.75, 119.06, 43.39, 40.33, 30.20, 20.39, 13.84. HRMS (ESI negative) calcd for [M-H] - 343.0855, found 343.0845. 3. General procedure for monitoring carboxylesterase activity. Based on the procedure following, all the measurements of carboxylesterase activity were carried out in 100 mM phosphate buffer (PBS, pH=7.4).With a total incubation volume of 0.2 mL, culture mixtures which contained PBS and mentioned enzymes protein or microsomes, were mixed gently. Then a proper amount of NCEN was added to start the reactions (contain 1% DMSO). After incubation at 37 °C in a shaking water bath for 60 min, equal volume of ice-cold acetonitrile was added to terminate the reactions. The mixtures were then centrifuged for 5 minutes at 20,000 g. Aliquots of supernatant were taken for further fluorescence analysis (Gain 80). In order to ensure that metabolites formation was enzyme dependent, control incubations were carried out without enzyme sources at the same time. All assays were performed in duplicates. 4. Cell culture and confocal fluorescence imaging. HepG2 cells were grown in MEM/EBSS culture medium (contain 10% FBS). Cells were seeding at a density of 1 × 105 cells per dish (Φ 20 mm) and incubated in a humidified incubator containing 5% CO2 at 37 °C overnight. The adherent cells were washed twice with FBS-free culture medium and incubated with/without 100 μM LPA (preparing in FBS-free culture medium) for 30 min at 37 °C in 5% CO2 incubator. The stock solution of probe NCEN (20 mM) in DMSO was diluted into the cell culture media (FBS free) to a final concentration of 20 μM. The cells were then incubated at 37 °C for another 60 min, followed by rinsing with PBS (pH 7.4) for three times to remove the extracellular probe, and imaged under confocal microscope (Olympus, FV1000). In one photon microscopy assay, excitation wavelength was set at 405 nm, blue emission was collected with a 430-480 nm window, and


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Scheme 1 Schematic illustration of NCEN and its fluorescence response toward hCE2.

Fig 1. Time course of absorption spectra of NCEN (10 μM) upon addition of hCE2 at 37 °C. Spectroscopic properties were recorded in PBS-CH3CN (pH = 7.4, 1:1)

green emission was collected with a 520-570 nm window. In two-photon mode, images were acquired under 800 nm excitation and fluorescent emission windows of 420460 nm (blue) and 495-540 nm (green). 5. Preparation of fresh mouse liver slices and confocal fluorescence imaging. Slices were prepared from the liver of 7-day-old mice. Slices were cut to 100 μm thickness by a vibrating-blade microtome and placed into glass-bottomed dish. For the fluorescence imaging experiments, slices were incubated with 20 μM NCEN in PBS buffer bubbled with 95% O2 and 5% CO2 at 37 ˚C for 1 h. Slices were then washed three times with PBS, and observed under a two-photon confocal microscope (Olympus FV1000). RESULTS AND DISCUSSIONS

1. Synthesis and sensing mechanism. Suitable fluorescent probes for in vivo hCE2 detection should own certain characteristics, such as two-photon absorption, ratiometric fluorescent, fast and sensitive response, and good selectivity. Meanwhile, a fluorescent probe for hCE2 should consist of two elements, a detecting part and a fluorescence reporting part. In this work, we employed NAH as two-photon fluorophore for its desirable spectroscopic properties and feasibility in structural modification.34-39 Probe NCEN was constructed by the incorporation of a chloracetyl into NAH via amide bond, which served as an efficient reaction site for hCE2. As anticipated, the ratiometric detection of hCE2, involved the reaction of NCEN with hCE2, the amide bond was cleaved, and the green fluorescence was stored (Scheme 1). The

Fig 2. Time course of fluorescence emission spectrum of NCEN (10 μM) upon addition of hCE2 in PBS-CH3CN (pH = 7.4, 1:1) when excited at 354 nm (a) and 430 nm (b), repectively. Emission color (under a 365 nm light) changes are inserted.

details of the synthetic procedure of NCEN are described in the Supporting Information. The chemical structures of NCEN were verified by HRMS, 1H NMR, and 13C NMR (see Fig S1-3 in the Supporting Information). 2. Spectral properties of NCEN towards hCE2. Firstly, the sensing ability of NCEN for hCE2 hydrolytic activity was investigated. As shown in Fig S4, NCEN is very stable in phosphate buffer (PBS) without any metabolite detected following 60 min incubation at 37 °C. In contrast, NCEN could be readily hydrolyzed and formed a single metabolite upon addition of hCE2 or hCE2 containing human tissue preparations, such as human liver microsomes (HLM) and human intestine microsomes (HIM). The formation of its hydrolytic product of NCEN was fully identified as NAH, based on the LC retention time, UV spectra and MS/MS spectra with the help of standard (see Fig S4 in the Supporting Information). To demonstrate the ratiometric feature of the NCEN/NAH system, the UV–vis absorption and fluorescence emission spectra of NCEN upon addition of hCE2 were depicted in aqueous medium at physiological pH. As shown in Fig 1, NCEN exhibited an absorption band at 354 nm in PBS– acetonitrile (v: v = 1: 1, pH 7.4 at 37 °C), the solution was colorless due to the strong electron-withdrawal effect of the chloracetyl moiety. In sharp contrast, upon addition of hCE2 (10 μg/mL) for 60 min, the UV–vis absorption around 354 nm decreased evidently, while a new absorpt-



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ion peak appeared at 430 nm. Such changes in absorption bring the solution from colorless to yellow, which could be clearly distinguished by “naked eyes”. Concomitantly, upon addition of hCE2, the maximum emission peak underwent a red-shift from 452 nm to 542 nm (Fig 2). The desirable red-shifted emission after hydrolysis should be attributed to the stronger ICT efficiency of the released amino compound NAH. More important, the ratios of emission intensities at 542 nm and 452 nm (I542/I452) werelinearly related to the incubation times up to 60 min (see Fig S5 in the Supporting Information). Therefore, further enzymatic kinetic analysis and quantitative determinations for hCE2-mediated NCEN hydrolysis were conducted within 60 min. In addition, we found the solvent DMSO had great interference on the catalytic activity of hCE2 (see Fig S6 in Supporting Information), hence, the final concentration of DMSO in all reaction mixture should not exceed 2 % (v/v). The fluorescence quantum yields of NCEN and NAH in PBS–acetonitrile (v: v = 1: 1) were also determined as 0.47 and 0.63, respectively, by using fluorescein as standard. These findings demonstrated that NCEN could serve as a highly desirable colorimetric and ratiometric fluorescent sensor for measuring the enzymatic activity of hCE2. Furthermore, it was found that the fluorescence intensities of NCEN and NAH were quite stable in a wide pH range from 1.5 to 11.0 (see Fig S7 in the Supporting Information). These properties are beneficial to the application of bioimaging in complex biological systems of which the pH values are varied in different subcellular compartments.40, 41 3. Selectivity of NCEN. The selectivity of NCEN towards hCE2 was then investigated under physiological conditions (pH 7.4 at 37 °C). A panel of human enzymes with hydrolytic activity was chosen to evaluate their possible interactions with NCEN (Fig 3). To our delight, only hCE2 can trigger a markedly increase in the fluorescence intensity at 542 nm and dramatic decrease at 452 nm when NCEN (10 μM) was incubated with selected enzyme. In sharp contrary, other hydrolytic enzymes including hCE1b, hCE1c, а-chymotrypsin (а-CT), pepsin, trypsin, lysozyme, carbonic anhydrase I (CA), а1-acid glycoprotein (AAG), dipeptidyl peptidases (DPP-IV, DPP-VIII and DPP-IX), fibroblast activation protein (FAP), bovine serum albumin (BSA), human serum albumin (HSA), cholinesterases (AChE and BChE) and paraoxonases (PON1 and PON2) could not bring evident changes in fluorescence spectra. Upon addition of 10 μg/mL hCE2, the ratio of the fluorescence intensity at 542 and 452 nm was elevated nearly 80-fold when it was compared with the control samples (without hCE2). Moreover, the fluorescence response of NCEN to various biologically relevant small molecules was also investigated to further explore its antiinterference ability in complex biological systems. As shown in Fig S8, the unique fluorescence performance of NCEN towards hCE2 could not be influenced upon addition of common biological metallic ions or amino acids in human tissues or fluids. All these results clearly suggested

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Fig 3. Fluorescence intensities ratio (I542/I452) of NCEN (10 μM) upon addition of different hydrolases. The spectra were measured in PBS-acetonitrile (v: v = 1: 1, pH 7.4) at 37 °C for 90 min. λex = 354/430 nm

Fig 4. The inhibitory effects on the hydrolysis of NCEN with use of a series of CEs inhibitors. All of the inhibitors applied were at 100 μM. The spectra were measured in PBSacetonitrile (v: v = 1: 1, pH 7.4) at 37 °C for 60 min. λex = 354/430 nm.

that NCEN displayed excellent selectivity for hCE2 over other biologically relevant species and could be of value for highly selective sensing of hCE2 in complex biological samples. To further confirm that the fluorescence changes in complex biological systems were hCE2 dependent, chemical inhibition assays were conducted in human tissue preparations by using a series of selective esterase inhibitors. As shown in Fig 4, bis(4-nitrophenyl) phosphate (BNPP, a potent inhibitor of carboxylesterases) and loperamide (LPA, a selective inhibitor of hCE2) could strongly inhibit the hydrolysis of NCEN, while other inhibitor including huperzine A (HA, a selective inhibitor of AChE) and ethylene diamine tetraacetic acid (EDTA, a selective inhibitor of PON) displayed negligible inhibitory effects. Moreover, both BNPP and LPA inhibited hCE2mediated NCEN hydrolysis in a dose-depended manner (see Fig S9 in the Supporting Information). These results further suggested that NCEN could act as a highly selective probe for high throughput screening and characterization of hCE2 inhibitors by using human tissue preparations as enzyme sources. 4. Enzymatic kinetics of NCEN. It is well-known that


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ACS Applied Materials & Interfaces wards hCE2 and ideal kinetic behaviors, which inspired us to use this probe reaction as an efficient tool for the quantitative measurement of hCE2 in human biological samples. 5. The Sensitivity and Detection Limit of NCEN. Next, the linear response ranges for hCE2 quantification using NCEN as probe substrate were evaluated under physiological conditions. Under the optimized incubation conditions (pH 7.4 at 37 °C for 60 min), the fluorescence signal changes of NCEN upon addition of hCE2 at different concentrations were monitored. As shown in Fig 6, upon progressive addition of hCE2, the peak band at 452 nm decreased while the fluorescence intensities at 542 nm gradually increased. Furthermore, the ratios of emission intensities at 542 nm and 452 nm exhibited an excellent linear relationship (R2 = 0.98) to the concentrations of hCE2 in the range from 0.5 to 10 μg/mL (see Fig S10 in the Supporting Information). The limit of detection (3σ/slope) of NCEN for hCE2 was also estimated as 12 ng/mL. Such sensitivity is highly enough to determine hCE2 in living cells and tissues.

Fig 5. Michaelis-Menten kinetic plot (a) and Eadie-Hofstee plot (b) of NCEN hydrolysis in HIM, HLM and hCE2. Catalytic activity of hCE2 was determined by use of the fluorescence intensity at 542 nm. The excitation wavelength was 430 nm.

Table 1. Kinetic parameters for NCEN hydrolysis in different enzyme sources. Enzyme sources

Vmax

Km

CLint

(nmol/min/mg)

(μM)

(μL/min/mg)

HIM

5.08±0.15

8.12±0.56

625.62

HLM

4.52±0.12

6.13±0.43

737.36

hCE2

34.87±0.99

8.58±0.55

4064.10

the kinetic behavior was crucial for the quantitative applications of the activity-based fluorescent probes.42 In this study, the enzymatic kinetics of NCEN hydrolysis was well-characterized in different enzyme sources including recombinant hCE2, HLM and HIM. NCEN hydrolysis in hCE2, HLM and HIM followed the classic Michaelis– Menten kinetics, which was evidenced by the corresponding Eadie–Hofstee plots (Fig 5). As shown in Table 1, NCEN hydrolysis in both hCE2 and human tissue preparations displayed high affinity (Km< 10 μM) and good reactivity (kcat/Km >500 μL/min/mg protein). Furthermore, NCEN hydrolysis in both hCE2 and human tissue preparations displayed very closed Km values (8.58 μM in hCE2, 8.12 μM in HIM, and 6.13 μM in HLM), implying that hCE2 was the predominant enzyme responsible for NCEN hydrolysis in human tissues. These results demonstrated that NCEN hydrolysis displayed excellent selectivity to-

6. Quantification of hCE2 in human liver microsomes. To further explore the practical applications of NCEN, we assessed the hydrolytic activities of hCE2 in a panel of twelve individual HLM samples by using NCEN as a probe substrate. As shown in Fig 7a, about 3.4-fold individuals differences in the catalytic activity of hCE2mediated NCEN hydrolysis were observed, which was in accordance with previous literature reported variability in hCE2 levels obtained from HLMs and the variability in enzymatic activity assayed by other hCE2 probe substrates.43-46 Moreover, a strong correlation with a high coefficient parameter (R2=0.97, P < 0.0001) was gained between the hydrolytic rates of NCEN and the hydrolytic rates of fluorescein diacetate (FD, a commercially available probe of hCE2) (Fig 7b). These findings strongly suggested that NCEN could be employed to measure the real activity of hCE2 in complex biological samples with multiple human enzymes, and the quantification was highly reliable. 7. Bioimaging of hCE2 in living cells and tissues. To further expand the potential applications of this newly developed probe in bioimaging related fields, especially cellular and tissue imaging, NCEN was intentionally applied for bioimaging of endogenous hCE2 in living cells and deep tissues. Prior to bioimaging, the two-photon absorption cross sections of NCEN and its hydrolytic product NAH were determined by two-photon induced fluorescence measurement technique. As shown in Fig 8, both NCEN and NAH exhibit excellent two-photon properties, which could be excited with near-infrared light (two-photon absorption). With the excitation at 800 nm, the two-photon action cross section (δ) values of both NCEN and NAH were much close (512 GM, and 542 GM, respectively). These results suggested that NCEN could be served as a two-photon ratiometric fluorescent probe for imaging of endogenous hCE2 in living specimens with



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Fig 6. The changes in fluorescence spectra of NCEN (20 μM) upon addition of increasing concentrations of hCE2 (0.5-10 μg/mL) in PBS–acetonitrile (v: v = 1: 1, pH 7.4) at 37 °C for 60 min. (a) λex = 354 nm; (b) λex = 430 nm.

Fig 7. (a) The hydrolytic activities of NCEN in a panel of individual HLM samples (n = 12). (b) Correlation analysis between the hydrolytic rates of FD and the hydrolytic rates of NCEN in a panel of individual HLM samples (n = 12). The spectra were measured in PBS-acetonitrile (v: v = 1:1, pH 7.4).

high resolution and sensitivity. Prior to cell imaging, MTT assay was carried out to investigate the cytotoxicity of NCEN and NAH. The results showed that both of NCEN and NAH exhibited relatively poorly cytotoxicity to living HepG2 cells, and the cell viability was more than 80% upon addition of NCEN (25 μM) or NAH (25 μM) at 37 °C for 48 h (see Fig S11 in the Supporting Information). In these cases, NCEN (20 μM) was co-incubated with HepG2 cells and the confocal fluorescence images were recorded both in one and two-photon modes. As shown in Fig 9, cell loaded with NCEN (20 μM) for 60 min at 37 °C showed an obvious strong green fluorescence (for NAH) and dim blue fluorescence (for NCEN) when excited at 800 nm. By contrast, when the cells pretreated with hCE2 inhibitor LPA (100 μM), and followed by incubation with NCEN, a significant fluorescence en hancement in blue-channel and a minor decrease in green emission were observed. Importantly, when pre-treated with a specific CE2 inhibitor and further incubated with probe, cells afforded an intense ratiometric response (blue to green, Fig 9 l). The consistent changes in fluorescence were also observed in one photon microscopy (see Fig S12 in the Supporting Information). These results suggested that NCEN was cell membrane permeable and

could be used for fluorescence imaging of hCE2 in living cells by both OPM and TPM. To further demonstrate the specific hydrolysis of NCEN by endogenous hCE2, chemical inhibition assays were conducted in HepG2 cells lysates. These findings demonstrated that the pretreatment of hCE2 inhibitor in lysates could remarkably reduce the fluorescence intensity ratio (see Fig S13 in the Supporting Information). All these results indicated that NCEN could be served as a specific probe for measuring the functions of endogenous hCE2 and exploring its related biological processes in living cells. To further assess the application potential of NCEN as a two-photon fluorescent probe for bioimaging of hCE2 in deep tissues, we next investigated the sensing ability of this probe for CE2 in fresh mouse liver specimen. Prior to tissue imaging, the specificity of NCEN towards CE2 in mouse liver was investigated firstly. As shown in Fig S14, both BNPP (a potent inhibitor of CEs) and LPA (a selective inhibitor of CE2) could strongly inhibit the hydrolysis of NCEN in mouse liver preparations, implying that CE2 in mouse liver is the key enzyme responsible for NCEN hydrolysis. In this case, NCEN was co-incubated with mouse liver slice in PBS (pH 7.4 at 37 °C) for 60 min, and subsequently washed with PBS three times to remove the


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ACS Applied Materials & Interfaces residual probe before confocal fluorescence imaging procedure. As shown in Fig 9, under excitation at 800 nm, TPM images showed strong fluorescence responses in green channel (495 - 540 nm) at a depth of 50 μm, indicating that CE2 are abundant in mouse liver. These data indicate that NCEN is capable of measuring endogenous CE2 in living tissues by TPM, which could be attributed to its good two-photon fluorescence properties and the increased penetration depth by TPM. CONCLUSION

Fig 8. Two-photon absorption cross-section of NCEN and NAH under 750–850 nm excitation light (measured in PBS/acetonitrile solution).

Fig 9. Two-photon confocal fluorescence imaging of NCEN (20 μM) stained with HepG2 cells. (a- d) HepG2 cells only. (e-h) Cells were incubated with NCEN (20 μM) at 37 °C for 60 min. (i- l) Cells were pre-treated with LPA (100 μM) and then added NCEN (20 μM) at 37 °C for 60 min. Images were acquired under excitation at 800 nm with two fluorescent emission windows: (b, f, j) blue = 420 – 460 nm, (c, g, k) green = 495 – 540 nm. (d), (h) and (l) displayed the ratio of emission intensities collected in optical windows between 420-460 (blue) and 495-540 nm (green). Scale bars = 10 μm.

In summary, a ratiometric two-photon fluorescent probe NCEN was developed and well characterized for highly selective detection of hCE2 in living cells and tissue for the first time. The newly developed probe displayed excellent selectivity towards hCE2 over other human hydrolases and biologically relevant species. NCEN could be readily hydrolyzed by hCE2 and brought remarkable changes in both colour and fluorescence spectrum, allowing the naked-eye and fluorescence analyses to distinguish obviously. Furthermore, both NCEN and NAH exhibit excellent two-photon properties, which could be excited with near-infrared light to avoid interference of biological matrix and photodamage to living sample. The successful biological applications of NCEN in living cells and tissues revealed that this probe could selectively detect endogenous hCE2 with high resolution and sensitivity in complex biological systems, with the help of TPM detection. All these findings suggested that NCEN could serve as a highly specific two-photon fluorescent probe for real-time monitoring of endogenous hCE2 in living cells and for exploring its associated biological functions in complex biological systems. ASSOCIATED CONTENT Supporting Information Detailed experimental procedures and structural characterization of compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author [email protected]; [email protected] & [email protected] Author Contributions d These authors contributed equally Notes The authors declare no competing financial interests. ACKNOWLEDGMENT Fig 10. Two-photon confocal fluorescent images of endogenous CE2 in a mouse liver slice stained with NCEN (20 μM). Images (60× magnification) were acquired at depth within 50 μm with 800 nm excitation and fluorescent emission windows of 420 - 460 nm and 495 - 540 nm. Scale bars = 30 μm.

This work was supported by the National S&T Major Projects of China (2012ZX09501001, 2012ZX09506001), the National Basic Research Program of China (2013CB531800), NSF of China (81473181, 81302793, 21421005 & 21572029), and the State Key Laboratory of Fine Chemicals (KF1304 and KF1408).



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