Near-Infrared Fluorescent Probe with Remarkable Large Stokes Shift

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Near-infrared Fluorescent Probe with Remarkable Large Stokes Shift and Favorable Water Solubility for Real-time Tracking Leucine Aminopeptidase In Living Cells and In Vivo Wenda Zhang, Feiyan Liu, Chao Zhang, Jian-Guang Luo, Jun Luo, Wenying Yu, and Lingyi Kong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03332 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017

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Analytical Chemistry

Near-infrared Fluorescent Probe with Remarkable Large Stokes Shift and Favorable Water Solubility for Real-time Tracking Leucine Aminopeptidase In Living Cells and In Vivo Wenda Zhang, Feiyan Liu, Chao Zhang, Jian-Guang Luo, Jun Luo, Wenying Yu*, and Lingyi Kong* Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China Abstract: Leucine aminopeptidase (LAP) is a kind of proteolytic enzymes and associated closely with pathogenesis of cancer and liver injury. Accurate detection of LAP activity with high sensitivity and selectivity is imperative to detect its distribution and dynamic changes for understanding LAP’s function and early diagnosing the disease states. However, fluorescent detection of LAP in living systems is challenging. To data, rarely fluorescent probes have been reported for imaging LAP in vivo. In this study, a novel probe (TMN-Leu) was developed by conjugating a near-infrared dicyanoisophorone derivative ﬂuorophore with LAP activatable L-leucine amide moiety for the first time. TMN-Leu featured large Stokes shift (198 nm), favorable water solubility, ultrasensitive sensitivity (detection limit of ~0.38 ng/mL), good specificity, excellent cell membrane permeability, low toxicity and a prominent near-infrared emission (658 nm) in response to LAP. TMN-Leu has been successfully applied to track LAP of cancer cells and normal cells, monitor LAP changes in different disease models and rapidly evaluate LAP inhibitor in cell-based assay. Notably, this probe firstly revealed that HCT116 cells with higher LAP activity were more invasive than LAP siRNA transfected HCT116 cells, suggesting that LAP might serve as an indicator reflecting the intrinsic invasion ability of cancer cells. Finally, TMN-Leu was also employed for in vivo real-time imaging LAP in living tumor-bearing nude mice with low background interference. All together, our probe possesses potential value as a promising tool for diagnostic application, cell-based screening inhibitors and in vivo real-time tracking enzymatic activity in preclinical applications.

INTRODUCTION Leucine aminopeptidase (LAP) is one of the important proteolytic enzymes.1 It belongs to the M1 and M17 peptidase families, which can cleave N-terminal leucine residues from substrates.2 Importantly, LAP is involved in various physiological and biological processes ranging from tumor cell invasion, proliferation, drug resistance, angiogenesis to liver injury.3-5 Therefore, in situ dynamic monitoring and indentifying intracellular LAP is imperative for LAP-related pathophysiology elucidation and disease diagnosis. Molecular bioimaging of enzyme activity in vitro and in vivo has been emerging as a powerful strategy for accurate disease diagnostics.6-15 The development of “smart” noninvasive imaging probes for the detection of specific enzyme activity in vivo is urgently required for cancer diagnosis and related disease detection.16-18 However, the complexity of dynamic living system makes it difficult to trace and visualize enzyme activity in vivo. In past years, fluorescent spectroscopy, a noninvasive imaging technique, has increasingly attracted interest attributed to its excellent advantages of high sensitivity, good selectivity, operation simplicity and in situ response. It has been applied in many fields such as biological monitoring and clini-

cal diagnosis.19-22 For detection of LAP activity in living cells, fluorescent probes have been attracted much more attention as well. However, it is still challenging to monitor the trace of the intracellular LAP with existing LAP probes because of the insufficient sensitivity (detection limit > 1 ng/mL), and some of the probes cannot be applied in living systems due to the short emission wavelengths and short Stokes Shift (Table S1). 9, 17, 23-25 To data, only one LAP probe was used for in vivo imaging, but its relatively small Stokes Shift limited further biological application.25 The NIR fluorphores have been paid increasing attention for in vivo imaging application with avoided interference from auto-fluorescence of indigenous biomolecules and higher penetrability to biological systems. While the large emission wavelength of NIR probes always with conjugated system of polycyclic aromatic rings leads to poor water solubility, and the high proportion of dimethyl sulphoxide or other organic solvent in test system may cause enzyme degeneration. The above-mentioned concerns encouraged us to develop novel NIR fluorescent LAP probes with large Stokes Shift, high sensitivity and good water solubility. In this work, a novel NIR emission fluorescent probe TMNLeu with large Stokes Shift and favorable water solubility was successfully developed. In our design, the L-leucine amide

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moiety was chosen as the enzyme active trigger and the dicyanoisophorone group was utilized as the NIR chromophore due to its advantages of excellent membrane permeability, applicable fluorescence and especially the NIR emission wavelength. Which in turn allows for in vivo real-time imaging of LAP activity in colorectal tumor-bearing nude mice. The enzyme-triggered amide moiety of TMN-Leu was cleaved upon interaction with a suitable LAP, resulting in a distinct turn-on broad emission fluorescence signal at the NIR region. TMN-Leu is a high sensitivity and selectivity LAP probe with excellent features such as low cytotoxicity, light-up NIR emission, ultrasensitive response, large Stokes shift, favorable water solubility, real-time and high signal-to-noise ratio in bioimaging of trace LAP in living cells and in vivo. To the best of our knowledge, the NIR light-up TMN-Leu is the first mitochondria-targetable fluorescence probe for real-time in vivo tracking of LAP activity in tumor-bearing nude mice. EXPERIMENTAL SECTION Materials and Methods. Unless special stated, all chemicals and solvents including 4-nitrobenzaldehyde, petroleum ether, isophorone, acetonitrile, dichloromethane, malonitrile, ethyl acetate, was supplied by Sinopharm chemical reagent Co. Ltd., Nitroreductase, GGT, Monoamine oxidase A (MAOA) and Leucine aminopeptidase (LAP) were obtained from Sigma-Aldrich Co. Ltd. 1

H NMR spectra were measured using a Bruker spectrometer (500 MHz) and referenced to TMS. HRMS analysis was performed on a Mariner ESI-TOF spectrometer. All pH measurements were measured with a sartorius basic pH-meter PB10. HPLC analysis was obtained on an Agilent 1100 series. The UV-visible absorption spectra were performed on a Shimadzu UV-1700 spectrometer and the fluorescence spectra were performed on a Hitachi F-7000 luminescence spectrometer respectively or Multi-Mode Detection Platform. Cell imaging was observed under an ImageXpress Micro Confocal analysis. In vivo imaging was obtained using a CRI Maestro small animal in vivo imaging system. Synthesis. LAP activatable NIR fluorescence imaging probe TMN-Leu was synthesized according to the procedure in Scheme S1, and its structure characterization were described in the Supporting Information. General Procedure to Monitor LAP Level in Vitro. All spectrum measurements were performed in PBS buffer (10 mM, pH 7.4) at 37 °C. The stock solution of probe TMN-Leu (10 mM) was prepared in DMSO. Various physiologically important species (Ca2+ , Fe3+ , Mg2+, Mn2+, H2O2, GSH, Cys, Hcy, MAO-A, GGT, Nitroreductase) were prepared in doubledistilled water. The test solution was prepared by mixing the calculated amount of probe into PBS to obtain a final concentration of 10 µM. LAP and other analytes were also dissolved in test solution with an appropriate concentration. The UV-vis absorption spectra data was recorded from 300 to 550 nm, and the fluoresecence emission spectra was collected in the range from 550 to 750 nm (λex= 460 nm, λem= 658 nm, slit widths: 5 nm/5 nm) after incubation for 70 min. Cell Culture and Cytotoxicity Assay. HCT116 cells, L02 cells, MCF-10A cells, HepG2 cells and MDA-MB-231 cells were provided by Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China), and were grown in DMEM medium supplemented

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with 10% fetal bovine serum at 37 °C in a 5% CO2/95% air incubator. The cytotoxicity of the probe to HCT116 cells and HepG2 cells was evaluated through MTT assays. The cells were initially seeded into 96-well plate at a density of 6×103 cells/well, and various concentrations (0, 2.5, 5.0, 10, 20 µM) of the probe in 200 µL DMEM mediums were added into each well. After incubation at 37°C in a 5% CO2/95% atmosphere for 24 h. 0.02 mL of the MTT (0.5 mg/mL) were diluted in the plates and followed by culture for another 4 h. After treatment, the formed formazan were dissolved after placing 150 µL DMSO in the wells. Then, the cell viability rate was calculated by measuring the absorbance values at 570 nm with a reference wavelength at 630 nm by ELISA reader (Spectra Max Plus 384, Molecular Devices, Sunnyvale, CA). Fluorescence Microscopy Imaging of LAP in Cells. Initially, cells were seeded into a 96-well plate and cultured in DMEM medium supplemented with 10% FBS in an atmosphere of 5% CO2 at 37°C. Before imaging, 100 µL of media containing 10 µM of TMN-Leu was added to the wells and incubated. After treatment, the images of cells were taken under an ImageXpress Micro Confocal analysis. For Cisplatin stimulation, a concentration of Cisplatin (2 mg/L) solution was added to the 96-well plates containing adherent cells in DMEM medium contained with 10% (v/v) FBS at 37°C in a humidified 5% CO2 incubator, and the cells were incubated for 12 h. For Ace (acetamidophenol) stimulation, an appropriate concentration of Ace solution was added to the 96-well plates containing adherent cells in DMEM supplemented with 10% (v/v) FBS at 37°C in a humidified 5% CO2 incubator, and the cells were incubated for 48 h. For Bestatin inhibition, Bestatin (100 µM) solution was pretreated to the 96-well plates containing adherent cells in DMEM supplemented with 10% (v/v) FBS at 37°C in a humidified 5% CO2 incubator before the probe was added, and the cells were incubated for 1 h. Evaluation of Inhibition Efficacy of Bestatin in Living Cells. To evaluate the inhibitory efficacy of Bestatin towards LAP in cell-based assay, HCT116 cells were firstly treated with Bestatin with concentration ranges from 0-100 µΜ for 1 h. Then, the medium was removed and the cells were washed with PBS for three times. After that, cells were incubated with TMN-Leu (10 µM, 100 µL) for another 65 min. The fluorescence images were taken on an an ImageXpress Micro Confocal analysis without washing operation. Each cell was taken as a region of interest, and fluorescence intensities were evaluated and averaged (Fluorescence intensities were measured in 3 regions in each well). Trans-well Invasive Assays. Cells were seeded at 2 x 104 cells per well (0.2 mL) and allowed to grow for 24 h. After being fixed in 4% of paraformaldehyde for 10 min. Crystal violet solution (Sigma, St. Louis, MO, USA) was used to stain the colonies for 4 h. The migration of cells was visualized at x200 magnification using a Leica Microscope. Fluorescence Imaging and Colocalization Studies. Initially, cells were seeded into a 96-well plate and cultured in DMEM medium supplemented with 10% FBS in an atmos-


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phere of 5% CO2 at 37 °C. After 24 h, 100 µL of fresh media containing 10 µM of TMN-Leu was added to the wells and incubated for 65 min. After treatment, the medium was removed, and the cells were washed with PBS for three times. Cells were then incubated with 1 µM Mito-Tracker Green or 1 µM Lyso-Tracker Green for another 30 min. After being fixed in 4% of paraformaldehyde for 10 min. The images of cells were taken under an ImageXpress Micro Confocal analysis.

cence intensity increase (Figure 1C), indicating that the enzyme-triggered cleavage reaction led to the release of free TMN-NH2. Together, this probe could be applied as a “turnon” fluorescent sensor which possessed a sensitive response to LAP. More importantly, the long-wavelength emission of the probe toward LAP with an obvious large Stokes shift (198 nm) was highly favorable for bioimaging analysis and this efficiently decreased the measuring error induced by the excitation light and scattered light.

In vivo Imaging of LAP Activity in Tumor-bearing Nude Mice. All Animal procedures were performed in accordance with the requirements of the Animal Ethics Committee of China Pharmaceutical University, and the guidelines of the National Institutes of Health. All animal experiments were approved by the Animal Ethics Committee of China Pharmaceutical University. For in vivo imaging, 5 weeks old BALB/c nude mice were selected and inoculated with HCT116 cells on the auxiliary region by injecting 3×106 cells subcutaneously to establish tumor model. Tumors were allowed to grow to 8-12 mm in diameter and then used for experiment. To restrain the endogenous LAP activity, the tumor region was given a subcutaneous injection with or without Bestatin (100 µM, 50 µL) and incubate for 1 h, subsequently, the probe TMN-Leu (50 µM, 50 µL) was injected subcutaneously into tumor region of the tumor-bearing nude mice. The in vivo imaging was obtained by using a CRI Maestro small animal in vivo imaging system, with excitation of red filter (460 nm~560 nm). RESULTS AND DISCUSSION Design and Synthesis. The NIR fluorphores have been paid increasing attention for in vivo imaging application with avoided interference from auto-fluorescence of indigenous biomolecules. Moreover, due to their higher penetrability to biological samples compared with the shorter wavelength, the dicyanoisophorone derivatives were used as an NIR-active fluorophores to detect biomarker for disease diagnosis and pathophysiology elucidation. Here, we envisioned that the merge of the L-leucine moiety and a fluorophore TMN-NH2 could quench the emission intensity of the probe by the strong electron-withdrawing amide bond. After treated with LAP, the free TMN-NH2 was specially activated and the electron-rich amine moiety was released, then yielded a remarkable fluorescence signal (Figure 1A). To test our hypothesis, the probe TMN-Leu was designed, synthesized and confirmed by 1 HNMR, 13C NMR and HRMS. All the intermediates were also characterized by 1HNMR, 13C NMR and HR-ESI-MS (Scheme S1 and Figure S13-S23). Spectroscopic Properties and Optical Response to LAP. To investigate the ability to detect LAP, the spectral character of this probe responding to LAP was initially measured in the buffer solution (PBS/DMSO = 999:1, v/v, 10 mM, pH = 7.4), implying its favorable water solubility. As Figure 1B illustrated, the probe exhibited a faint yellow color and a maximum absorption wavelength at 395 nm, but upon the addition of LAP (165 ng/mL), the absorption maximum decreased, a distinctive increase of the absorption band centered at 460 nm could be clearly visualized with the solution color changed to pink. Simultaneously, the fluorescent spectra of probe TMNLeu treated with LAP was also systematically investigated, the maximum emission at 658 nm had a significant fluores-

Figure 1. (A) Proposed sensing mechanism of TMN-Leu for LAP enzymatic activation. (B) Absorption spectra of TMN-Leu (10 µΜ) before and after incubation with LAP (165 ng/mL) in PBS buffer (10 mM, pH 7.4, 0.1% DMSO). (C) Fluorescence response of 10 µΜ TMN-Leu (λex = 460 nm, λem = 658 nm) after incubation with various concentrations of LAP (0-330 ng/mL) for 70 min in PBS buffer (10 mM, pH 7.4, 0.1% DMSO) at 37oC. (D) Line relationship between the fluorescence intensity of TMN-Leu (658 nm) and the concentration of LAP (0.4-14.0 ng/mL). Error bars represent standard deviation (n = 3).

The time-dependent fluorescence response of the probe toward LAP was tested. As shown in Figure S1, the fluorescent intensity at 658 nm increased rapidly in a time-dependent manner, and then obtained a maximum intensity in approximately 70 min. Subsequently, the fluorescence intensity of the probe was varied upon addition of different concentrations of LAP (0-330 ng/mL) which was examined and recorded in Figure 1C, the continuous increase of LAP resulted in an obvious fluorescence intensity enhancement (658 nm), and reached a plateau at 165 ng/mL of LAP. Moreover, the fluorescent signal intensities were linearly proportional to LAP at the concentration range of 0.4-14.0 ng/mL (R2 = 0.9972, Figure 1D), the detection limit of TMN-Leu (3σ/slope method) was calculated to be as low as 0.38 ng/mL. This may enable the probe to trace amounts of intracellular LAP. In addition, the kinetic parameters of probe toward LAP was also determined by the Lineweaver-Burk plot, 1/V (reaction rate) versus 1/concentration (Figure S2). The Michaelis constant (Km) of the probe was estimated to be 24.1 µM with the MichaelisMenten equation, the calculated small Km indicating the probe possessing a strong binding affinity with LAP. Moreover, we also evaluated the fluorescence profiles of TMN-Leu verses different pH values to investigate its practi-



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cability for biological imaging (Figure S3). No obvious emission was changed at 658 nm above the pH range of 5-9, indicating the stable structure of the probe during acidic to alkaline conditions. As expected, upon addition of LAP (165 ng/mL), the emission intensity increase was sensitive to the pH range from 5-9, especially remained the maximum intensity at the physiological pH window. The results indicated that the probe had obvious advantage and highly practical value for imaging application in biological systems. Figure 3. (A) Fluorescence response of TMN-Leu (10 µΜ) toward LAP (165 ng/mL) in the absence and presence of Bestaint at concentration of (a) 0, (b) 5, (c) 15, (d) 30 µΜ for 70 min in PBS buffer (10 mM, pH 7.4, 0.1% DMSO) at 37 oC. Error bars represent standard deviation (n = 3). (B) Fluorescence response of TMN-Leu (10 µΜ) and TMN-NH2 in the absence of Bestatin (530 µΜ) for 70 min in PBS buffer.

Figure 2. Fluorescence spectra of TMN-Leu (10 µΜ) upon interaction with LAP or other analytes in PBS buffer (10 mM, pH 7.4, 0.1% DMSO). Columns 1-9: PBS, Ca2+, Fe3+, Mg2+, Mn2+, H2O2, GSH, Cys, Hcy; 10-12: MAO-A, GGT, Nitroreductase. 13: LAP. All measurements were taken at 37oC. Error bars represent standard deviation (n = 3).

To further confirm the fluorescence monitoring specificity of TMN-Leu toward LAP, the fluorescence signal effect of the probe to other species (including 10 equivalent of Ca2+, Fe3+, Mg2+, Mn2+, H2O2, GSH, Cys, Hcy, 10 U/L of MAO-A, GGT, Nitroreductase) were detected in the PBS buffer solution (0.1% DMSO, 10 mM, pH = 7.4). As Figure 2 illustrated, the addition of other species exhibited a negligible fluorescence response, only LAP could induce a remarkable fluorescence enhancement. Furthermore, the addition of these chemical species had little effect on the proteolytic activity of LAP (Figure S4). All data revealed that TMN-Leu exerted excellent specificity under physiological conditions. To gain more insights into the specificity of the probe toward LAP, a well-known LAP inhibitor (Bestatin) was pretreated with LAP before treated the enzyme with TMN-Leu (10 µM). As shown in Figure 3A, Bestatin could efficiently suppress the fluorescence response of LAP in a dosedependent manner, while it hardly affected the fluorescence of TMN-NH2 and TMN-Leu in the same concentration (Figure 3B). These data indicated that TMN-Leu could be applied as an excellent candidate for specific LAP detection in biosamples.

Enzyme-Catalytic Activation Mechanism. As aforementioned, the cleavage of L-leucine amide moiety by the enzymereactive reaction could liberate the electro-donating amino group accompanying by the generation of TMN-NH2. To verify the activation mechanism, HPLC analysis were used. As shown in Figure 4A, the chromatographic peak of TMN-NH2 and TMN-Leu were found at 11.08 min and 6.58 min respectively. Additionally, upon addition of LAP (165 ng/mL) and incubation with TMN-Leu for 30 min, the reaction product exhibited a chromatographic peak at 11.08 min which matched perfectly with compound TMN-NH2, a weak peak was also observed at 6.58 min, corresponding to probe TMN-Leu. Moreover, the product of the reaction of TMN-Leu and LAP was further examined by HRMS. The peak at m/z 290.1651 [M+H]+, corresponding to TMN-NH2 and at m/z 403.2494 [M+H]+ corresponding to TMN-Leu were clearly observed (Figure 4B), indicating the evolution of TMN-NH2 upon the enzyme-triggered cleavage reaction. These data confirmed the fact that TMN-Leu could reacted with LAP to form TMNNH2 which emitted at NIR region with a distinct fluorescence signal.

Figure 4. (A) HPLC analysis of TMN-Leu (a) the reaction product of TMN-Leu by LAP (b) and TMN-NH2 (c). (B) HR-ESI-MS of TMN-Leu and (C) the reaction product of TMN-Leu by LAP.

In Situ Tracking of Endogenous LAP Activity in Living Cells. Considering the excellent sensing properties of the probe in vitro system, we further studied its capability of tracking LAP in living cells. Before cellular application, the biocompatibility of the probe was evaluated with two cell lines by MTT assays (Figure S5), the viability of HCT116 and HepG2 cells was almost 95% after incubation with a high concentration of 20 µM for 24 h, confirming the ultra low cytotoxicity of the probe to cells.


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We further applied the probe to assess the applicability of probe toward intracellular LAP in HCT116 cells under cultural condition using an ImageXpress Micro Confocal analysis. The experimental results were illustrated as Video S1 (cell images in Living Cell Station) and Figure S6, after addition with the probe 10 µM, a gradually increasing NIR fluorescence signal of living cells was investigated in a time-dependent manner. Through the Video S1, we found that the fluorescence signal reached a plateau after incubated with TMN-Leu for 65 min, which was almost consistent with in cell free LAP activity test, indicating good cell permeability of the probe and its reaction with the intracellular LAP in cells. This is the first time for the LAP probe to in situ image LAP activity in living cells under cultural condition. Meanwhile, the same conclusion was obtained on MDA-MB-231 cells (Figure S7). Next, we investigated the response of the probe at the different concentrations of TMN-Leu in HCT116 cells for 65 min. As Figure 5 illustrated, the fluorescence signal was increased in a good dose-dependent manner, which was further confirmed using flow cytometry (Figure S8). LAP was always overexpressed in cancer cells compared with normal cells. To further investigate the difference LAP content between cancer cells and normal cells, cancer cells HCT116 (Hoechst 33342 pre-stained) and normal cells MCF-10A were co-cultured and their LAP activity was detected by TMN-Leu, the fluorescence signal intensity in HCT116 was much stronger than that in MCF-10A cells (Figure S9), indicating the LAP in cancer cells HCT116 was more active than that in normal cells MCF10A. The above data showed that probe TMN-Leu possessed striking capability of tracking LAP in living cells and revealing the different LAP activity between cancer cells and normal cells.

therapy. To investigate the inhibitory effect of LAP inhibitor on the LAP activity in cells, the currently common detection methods such as western blot and RT-PCR employed in vitro need complex manipulation process and have relative low sensitivity. The fluorescence probe with high sensitivity, simple operation and excellent specificity may provide a concise method to detect LAP in situ. Subsequently, we investigated whether TMN-Leu could test the inhibition efficacy towards LAP activity using a fluorescence imaging experiment. HCT116 cells were pretreated with various concentrations of Bestatin for 1 h, following by incubation with TMN-Leu for another 65 min. The resulting fluorescence images were directly recorded without washing and fixing. As shown in Figure 6A, the fluorescence intensity decreased stepwise with the increasing concentration of Bestatin. To transfer the change of fluorescence intensity into I/I0 (I and I0 respectively represent the fluorescence data of inhibitor treated and untreated cells), we plotted the inhibitory histogram to give the IC50 value (concentration for half maximal inhibitory fluorescence intensity) to be ~5.7 µM (Figure 6B). Thus, TMN-Leu may have a great potential to be applied for the high-throughput cell-based assay to rapidly screen and evaluate new LAP inhibitors.

Figure 6. Fluorescence imaging-based LAP inhibitor assay with TMN-Leu. (A) Fluorescence images of HCT116 cells treated with LAP inhibitor Bestatin at concentration of (a) 100, (b) 50, (c) 25, (d)12.5, (e) 6.25, (f) 3.125 and (g) 0 µΜ for 1 h, and then incubated with TMN-Leu (10 µM) for another 65 min. (h)-(n) Differential interference contrast (DIC) images of the above corresponding cells. The images were captured without washing. Scale bar: 20 µm. (B) Plot of the change in intracellular fluorescence intensity (I/I0) versus Bestatin concentration based on the fluorescence images. Fluorescence intensities were evaluated at three regions in each dish. Error bars represent standard deviation (n = 3). The IC50 values of Bestatin for LAP in culturing HCT116 cells was found to be ~5.7 µM. Figure 5. Fluorescence imaging of endogenous LAP in HCT116 cells using different concentrations of TMN-Leu. (A) Fluorescence images of HCT116 cells treated with TMN-Leu at concentration of (a) 0, (b) 1.25, (c) 2.5, (d) 5, (e) 10 and (f) 20 µΜ for 65 min. (g)-(l) Differential interference contrast (DIC) images of the above corresponding cells. The images were captured without washing and imaged using an ImageXpress Micro Confocal analysis. Scale bar: 20 µm. (B) Fluorescence intensities were evaluated at three regions in each dish. Error bars represent standard deviation (n = 3).

Evaluation of Inhibition Efficacy of Bestatin in Living Cells. LAP is also confirmed as an efficient target and increasing number of LAP inhibitors have been developed for cancer

In Situ Tracking of Endogenous LAP Activity in Cisplatin-induced Cells. Treatment cancer cells with Cisplatin causes an increase of LAP, and the increased LAP plays a role in intrinsic resistance of cancer cells towards Cisplatin.1 Next, the effect of Cisplatin on the intracellular LAP change in HepG2 cells was investigated by TMN-Leu. As shown in Figure S10, the fluorescence intensity in Cisplatin-treated cells was almost 2-fold increased compared with the untreated HepG2 cells. To confirm that the increased fluorescence enhancement response was derived from the elevated LAP enzyme-catalyzed cleavage reaction, a control enzyme inhibition experiment was further performed by treating the fluorescence-increased HepG2 cells with Bestatin. The cells treated



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with Bestatin exhibited a marked fluorescence signal decrease, clearly indicating that Bestatin can effectively inhibit the LAP activity induced by Cisplatin in living cells. In other words, the fluorescence of the HepG2 cells indeed arose from the catalytic reaction of LAP. Thus, this probe could detect the intrinsic resistance of cancer cells by monitoring their LAP changes. In Situ Tracking of Endogenous LAP Activity in Druginduced Hepatotoxicity Model. Human body will be exposed to a wide of toxic substance from environmental and pharmaceuticals (such as Ace ) in one’s lifetime. While the main detoxification organ liver is related to a complex enzymatic system. Among them, LAP was confirmed as an indicator of liver dysfunction. Next, we examined the capability of the probe to image the endogenous LAP in an Ace-induced human normal hepatocyte cell line (L02). As shown in Figure S11, when L02 cells were incubated with TMN-Leu, the fluorescence in the cells gradually increased. Moreover, under the same imaging condition the Ace-pretreated L02 cells exhibited a largely increased fluorescence as compared to the control, which was further suppressed by the LAP inhibitor Bestatin. In short, this fluorescence probe can be used for detecting the alteration of the intracellular LAP activity in liver injury diseases.

Figure 7. Fluorescence imaging of endogenous LAP in HCT116 cells and siRNA-transfected HCT116 cells and their relative invasion activity. (A) Fluorescence images of HCT116 cells (b), HCT116 cells transfected with LAP siRNA (d), (a) and (c) Differential interference contrast (DIC) images of the corresponding cells. The images were captured without washing and recorded using an ImageXpress Micro Confocal analysis. Scale bar: 20 µm. Fluorescence intensities were evaluated at three regions in each dish. Error bars represent standard deviation (n = 3). (B) HCT116 cells (a and c) and siRNA-transfected HCT116 cells (b and d) were seeded in transwell chamber and incubation for 24 h, then fixed by 4% paraformaldehyde and dyed by crystal violet and imaged under microscope (magnification, x200). (C) The relative mRNA level of LAP in HCT116 cells and siRNA-transfected HCT116 cells, ***p < 0.001.

The Relationship between LAP and Intrinsic Invasion Ability. It has been found that LAP is related to the invasive ability of cancer cells. However, it is indistinct whether the endogenous LAP plays a great role in the intrinsic invasive ability of colorectal cancer cells. Subsequently, we knocked down the LAP in HCT116 cells, the LAP activity and invasive ability of them were evaluated by probe TMN-Leu and

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transwell assays. The fluorescence in LAP knockdown HCT116 cells was lower than that in parent cells which exerted more invasive (Figure 7A and 7B). These data suggested a positive correlation between the intrinsic invasion ability of cancer cells and LAP activity. Furthermore, their LAP level was also confirmed by RT-PCR (Figure 7C). Together, LAP may act as a simple indicator to monitor the intrinsic invasive ability of cancer cells.

Figure 8. Intracellular localization of HCT116 cells incubated with TMN-Leu. Images of cells pretreated respectively with 10 µM TMN-Leu for 65 min and subsequently 1 µM Mito-Tracker Green (or 1 µM Lyso-Tracker Green) for 30 min. Red channel, probe fluorescence (a) and (d); Mito-Tracker, Green channel (b); Lyso-Tracker, Green channel (e); the overlap (c) and (f); (g)-(h) Differential interference contrast (DIC) images of the corresponding cells. Scale bar: 20 µm.

Fluorescence Imaging and Colocalization Studies. Having performed the LAP imaging in cells, we proceeded to investigate the detected distribution of LAP at subcellular levels by TMN-Leu using colocalization experiments in HCT116 cells. Cells were pretreated with 10 µM TMN-Leu for 65 min and subsequently 1 µM Mito-Tracker Green or 1 µM LysoTracker Green for another 30 min. Interestingly, the fluorescence of released TMN-NH2 in red channel overlapped exactly with that of Mito-Tracker Green in green channel but that of lysosome indicating that the enzyme cleavage was mainly occurred in mitochondria (Figure 8). Meanwhile, the same subcellular distribution of released TMN-NH2 can also be confirmed in MDA-MB-231 cells (Figure S12). Thus, this probe may provide an excellent tool for tracking mitochondrial LAP. In Vivo Real-time Tracking of Endogenous LAP Activity in the HCT116 Tumor-bearing Mice. In vivo real-time imaging offers a powerful tool for accurately diagnosing disease and suspicious lesions with valuable spatiotemporal precision. Almost, the current fluorescent probes for imaging LAP activity are not suitable for in vivo experiments because their emissions are not located in NIR region and short Stokes Shift, thus fail to penetrate deeper tissue and are disturbed by intrinsic auto-fluorescence background signals. Therefore, with the prominent performance of the LAP probe in cellular fluorescence imaging, we further attempted to examine the applicability of our probe in vivo real-time visualizing the endogenous LAP activity in the HCT116 tumor-bearing mice. Before in vivo imaging application, a drug tolerance test was performed. Ten ICR mice were respectively given TMN-Leu at 5 mg/kg (0.2 mL) by intraperitoneal injection in a single dose, and no obvious cytotoxicity was observed. Next, the probe was applied to inject into the tumor region in HCT116 tumorbearing mice, followed by fluorescence imaging using a small


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animal in vivo imaging system. As shown in Figure 9, a gradually increased fluorescence response was observed in tumor region in a time-dependent manner. The fluorescence signal rapid responded to LAP activity and reached the plateau at 60 min. In the other group, after subcutaneously injection of Bestatin in tumor region of nude mice for 1 h, the probe was injected into the tumor region, the region treated with Bestatin exhibited little fluorescence signal under the same condition, indicating the probe was catalyzed by LAP to release TMNNH2 in the tumor. The result demonstrated that our probe could be utilized as a prominent bioimaging tool for in vivo real-time detecting LAP activity.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. A comparison of the fluorescent probes for LAP detection, synthesis of probe TMN-Leu, time-dependent fluorescence intensity of TMN-Leu with LAP, Lineweaver-Burk reciprocal plot, fluorescence intensity at different pH conditions, fluorescence spectra of TMN-Leu with LAP affected by other analytes, the MTT assays, time-dependently fluorescence imaging of endogenous LAP in HCT116 cells, fluorescence imaging of LAP in MDA-MB-231 cells, fluorescence monitor using an BD flow cytometry, fluorescence imaging of LAP in co-cultural cells, fluorescence imaging of endogenous LAP in Cisplatin-induced HepG2 cells, fluorescence imaging of endogenous LAP in Aceinduced L02 cells, intracellular localization of MDA-MB-231 cells incubated with TMN-Leu, the spectrums of 1H NMR, 13C NMR and HR-ESI-MS. Supporting Information (PDF) Video S1 (avi)

AUTHOR INFORMATION Corresponding Author * Lingyi Kong: E-mail: [email protected]. Figure 9. In vivo real-time fluorescence imaging of endogenous LAP in HCT116 tumor-bearing mice. (Group A) In vivo real-time fluorescence imaging of endogenous LAP in HCT116 tumorbearing mice after tumor injection TMN-Leu (50 µM, 50 µL). (Group B) Tumor region were pretreated with Bestatin (100 µM, 50 µL) for 1h, then tumor injection subcutaneously with TMNLeu (50 µM, 50 µL). And the images were acquired 5 min, 15 min 30 min, 45 min, 60 min, 75 min and 90 min after injection. The images were excited with a 460-560 nm filter, and acquired with a 600-700 nm long-pass filter.

CONCLUSIONS In conclusion, we firstly designed a novel ultrasensitive NIR fluorescent probe based on dicyanoisophorone for monitoring endogenous LAP in mitochondria with a low detection limit of 0.38 ng/mL. The probe featured striking characteristics in terms of large Stokes shift, favorable water solubility, high specificity and sensitivity, well pH independency, good cell membrane permeability, and low cytotoxicity enabling the efficiently tracking of trace LAP activity in cells. We successfully applied the probe to track LAP of cancer cells and normal cells, monitor the LAP changes on Ace-induced liver injury model and Cisplatin-induced drug-resistant model, and in situ to rapidly evaluate the LAP inhibitors on cell-based assay. Moreover, we found a positive relationship of the intrinsic invasive ability of colorectal cancer cells and their LAP activity by probe TMN-Leu, implying that LAP may act as a simple indicator to reflect the intrinsic invasion ability of cancer cells. More importantly, the NIR emission fluorescence of the probe with fast response make it possible for in vivo real-time visualization of endogenous LAP in living tumor-bearing mouse model with negligible background interference. We believe that the probe could serve as a useful indicator for distinguishing LAP in complex living systems and the diagnosis of LAPassociated diseases.

* Wenying Yu: E-mail: [email protected].

Author Contributions All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research work was supported by National Natural Science Foundation of China (Grant No. 81673298 and No. 81402791), Natural Science Foundation of the Jiangsu Higher Education Institutions of China (Grant No. BK20140670), Jiangsu Shuangchuang Talents for Oversea Ph.D.s, Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX17_0731). Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63), Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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