Lysosome-Targeting Fluorogenic Probe for Cathepsin B Imaging in

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry .... (b) Illustration of the mechanism of action of probe 1 for flu...
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A lysosome-targeting fluorogenic probe for cathepsin B imaging in living cells Yuqi Wang, Jinbo Li, Liandong Feng, Jingfang Yu, Yan Zhang, Deju Ye, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03717 • Publication Date (Web): 15 Nov 2016 Downloaded from http://pubs.acs.org on November 15, 2016

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Analytical Chemistry

A lysosome-targeting fluorogenic probe for cathepsin B imaging in living cells

Yuqi Wang, Jinbo Li, Liandong Feng, Jingfang Yu, Yan Zhang,* Deju Ye* and Hong-Yuan Chen State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China Fax: 025-89681905 *E-mail: [email protected] *E-mail: [email protected]

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Abstract: Cathepsin B (CTB) is a lysosomal protease which has been recognized as a promising biomarker for many malignant tumors, and accurate detection of its activity is important in early diagnosis of cancers and predicting metastasis. Herein, we reported a lysosome-targeting fluorogenic small-molecule probe for fluorescence imaging of lysosomal CTB in living cancer cells by incorporating a CTB recognitive peptide substrate Cbz-Lys-Lys-p-aminobenzyl alcohol (Cbz-Lys-Lys-PABA) and a lysosome locating group morpholine. We demonstrated that the probe could be efficiently activated by CTB to generate ~73-fold enhancement in fluorescence under acidic lysosomal environment (pH 4.5-6.0), allowing for high sensitivity and specificity to detect CTB. Fluorescence imaging results showed selective accumulation and fluorescence turn-on in the lysosomes of cancer cells, which were capable of reporting on lysosomal CTB activity in cancer cells and normal tissue cells. This study highlights the potential of using lysosome-targeting group to design sensitive and specific fluorogenic probe for fluorescence imaging of lysosomal CTB in living cells.

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INTRODUCTION Precise detection of enzyme activity and its location within living cells provides vital opportunity to unravel enzyme function in biological process, which is essential for the diagnosis and treatment of diseases.1,2 A myriad of analytical methods capable of detecting different enzyme activity have been actively reported.3-5 Among them, fluorescence imaging with a fluorogenic probe is particularly attractive as its fluorescence is selectively activated by a target enzyme, whose catalytic ability could trigger continuous turnover of many molecules of probe. This could provide low background but signal amplification for sensitive and real-time measurement of various enzymes in living cells, such as β-galactosidase,6-8 matrix metalloproteinase (MMP),9-11 caspases,12-14 cyclooxygenase-2 (COX-2),15,16 and fibroblast activation protein-α (FAPα).17 Cathepsins such as cathepsin B (CTB), cathepsin D (CTD) and cathepsin L (CTL) are lysosomal proteases belonging to the papain family responsible for lysosomal protein degradation in cells.18,19 Of all the cathepsins, CTB is of significant importance due to its essential role in various biological and pathological processes.19,20 CTB is expressed as an inactive precursor, which is activated under acidic environment of lysosomes to initiate subsequent physiological responses including proteolytic degradation, antigen presentation and cell apoptosis.20 In addition, overexpression of CTB has also been found in various malignant tumors, where the elevated CTB activity contributes greatly to enhanced angiogenesis, invasion and metastasis of tumors.21 Therefore, CTB has been recognized as a

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promising biomarker for tumor progression and metastasis. Accurate detection of its location and activity is important in early diagnosis of cancers and predicting metastasis. In the past few years, a number of fluorescent probes for the detection and imaging of CTB have been developed.22-25 These probes include both activity and substrate-based probes whose fluorescence signals were produced upon either covalently binding to the active site of CTB to promote probe accumulation or cleavage of substrate to trigger fluorescence turn-on. As pioneering works, Bogyo et al. have explored irreversible inhibitors to design a series of quenched fluorescent activity-based probes which could covalently bind to either CTB or CTL and generate enhanced fluorescence, enabling noninvasive and whole-body imaging of CTB and CTL activities in tumors.26,27 These provided a great potential for profiling and localizing CTB and CTL in tumors, however, the resulting inhibition of activity upon interactions may perturb the enzymes’ function which would generate a small fluorescence turn-on ratio. Alternately, substrate-based probes that exploited the catalytic power of an enzyme of interest have been developed, showing improved cellular and in vivo sensitivity over activity-based probes for CTB detection. For example, Kim et al. have developed a substrate-based nanoprobe consisting of a self-quenched CTB cleavable peptide substrate and chitosan nanoparticles which showed high sensitivity to detect CTB activity in three different metastatic tumors in vivo.28 Urano et al. have reported another elegant approach to design a substrate-based small molecule fluorogenic probe with more than 200-fold increase in fluorescence

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upon activation, which could allow for real-time visualization of intraperitoneally disseminated tumors. However, this probe also showed cross-reactivity to CTL and thus posed a limitation for accurate detection of CTB activity.29 Recently, Phenix et al. have used Cbz-Lys-Lys-p-aminobenzyl alcohol (PABA) as a CTB recognition substrate to design a prodrug-inspired small molecule fluorogenic probe with improved specificity for CTB over other cysteine cathepsins.30 However, its potential for detailed cellular imaging are limited in terms of the fluorophore that emits blue fluorescence upon activation and the lack of lysosome targeting ability. Considering the active intracellular CTB mainly locates in the lysosomes,31,32 the design of new substrate-based small molecule probes capable of efficient delivery into lysosomes in tumor cells is necessary for accurate detection of intracellular CTB activity, but remains unexplored.

Herein, we reported a new lysosome-targeting small molecule fluorogenic probe (1) for fluorescence imaging of lysosomal CTB in living cells by incorporating a peptide substrate and a lysosome locating group into an amino luciferin scaffold (Figure 1a). We showed that probe 1 was fluorescence silent initially, but could be selectively hydrolyzed by the CTB under acidic lysosomal environment (pH 4.5-6.0) to liberate probe 2, resulting in strong green fluorescence distributing mainly in lysosomes. As a result, probe 1 exhibited good sensitivity and specificity for the lysosomal CTB, which was applicable for the discrimination of CTB overexpressed tumor cells from normal tissue cells with low CTB activity. EXPERIMENTAL SECTION

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General Materials and Methods. All chemicals were purchased from commercial suppliers and used without further purification. The 1H and

13

C NMR spectra were

acquired on a 400 MHz Bruker AVANCE III–400 spectrometer. Data for 1H NMR spectra are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), or br (broadened); coupling constants are reported as a J value in Hertz (Hz); the number of protons (n) for a given resonance is indicated nH, and based on the spectral integration values. Electrospray ionization mass spectroscopic (ESI-MS) analysis was conducted on a SHIMADZU-2020 LC/MS system. High-performance liquid chromatography (HPLC) was carried out on Thermo Scientific Dionex Ultimate 3000 with CH3CN/H2O (1‰ CF3COOH) as the eluents. The fluorescence spectra were measured with a Shimadzu RF-5301 PC spectrofluorometer with a 1 cm quartz cuvette. The UV-Vis spectra were measured with a Shimadzu UV-3600 spectrometer. Fluorescent microscopy images were acquired with a Leica TCS SP5 confocal laser scanning microscope and Olympus IX73 fluorescent inverted microscope. Synthesis. The preparation of probes 1, 2, 3 and 4 and characterization of them were described in the Supporting information. General Procedure for Fluorescence Measurement. Probes 1, 2, 3 and 4 were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions. These probes were then diluted with CTB assay buffer (50 mM MES, pH 5.5, 1 mM EDTA, 1 mM TCEP, 0.1% Triton X-100) to prepare solutions for fluorescence analysis. Typically, probe 1 (10 µM) in assay buffer was incubated with or without human recombinant

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CTB (1 µg/mL) at 37 °C for 30 min, and the fluorescence spectra in the solutions were recorded on a Shimadzu RF-5301 PC spectrofluorometer with excitation at 350 nm and emission wavelength range from 400 to 650 nm. Determination of Sensitivity toward CTB. The sensitivity toward CTB was investigated by incubating probe 1 (10 µM) in CTB assay buffer with varying concentration of CTB (0 – 0.5 µg/mL). The reaction solutions (100 µL final volume for each solution) in a 96-well black plate were kept at 37 °C, and the fluorescence intensities at 525 nm were immediately monitored on a ThermoFisher Scientific Varioskan Flash microplate reader with excitation at 350 nm. The intensity in each well was recorded every minute, and last for 25 min. Determination of Selectivity toward CTB. The selectivity toward CTB was investigated by incubating probe 1 (10 µM) in CTB assay buffer with 1 µg/mL human recombinant CTB, CTS, CTD, CTL or CTB pretreated with its inhibitor (CA-074-Me, 2 µg/mL). The reaction solutions (100 µL final volume for each solution) in a 96-well black plate were kept at 37 °C, and the fluorescence intensities at 525 nm were immediately monitored on a microplate reader with excitation at 350 nm. The intensity in each well was recorded every minute, and last for 40 min. Enzyme Kinetic Studies. The KM,kcat and kcat/KM parameters for each compound were obtained by incubating different concentrations of probe with a constant concentration of activated enzyme. Briefly, a series of different concentrations of Cbz-Arg-Arg-AMC (10, 20, 40, 60, 80, 100 and 120 µM) in CTB assay buffer in a 96-well black plate were pre-warmed at 37 °C for 10 min, and the reaction was

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initiated upon addition of CTB (0.1 µg/mL) . CTB assay buffer was added to adjust the total volume to 100 µL each well. The fluorescence intensity at 460 nm was measured in a microplate reader (λex = 360 nm) at 37 °C every 30 seconds. The amount of AMC in each well was determined by a standard curve under the same conditions. The values of the KM and kcat were determined from the Lineweaver-Burk plot. Cell Cultures. HeLa, MDA-MB-231 or U87 cancer cells and HEK-293 normal tissue cells were cultured in high-glucose DMEM (Gibco) medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in 5% CO2 humidified atmosphere at 37 °C. KB cancer cells were cultured in high-glucose RPMI-1640 (Gibco) medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in 5% CO2 humidified atmosphere at 37 °C. 3T3 normal tissue cells were cultured in high-glucose DMEM (Gibco) medium containing 10% calf serum and 1% penicillin/streptomycin in 5% CO2 humidified atmosphere at 37 °C. MTT Assays. Approximately 5000 cells (HeLa or MDA-MB-231) were seeded in a 96-well plate and allowed growing in DMEM medium containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin overnight. The medium was removed, and the cells were then washed with serum-free DMEM medium. A series of different concentrations of probe 1 or 3 (10, 20, 40, 60 or 80 µM) in 100 µL of FBS-free DMEM medium were added and incubated for 24 h. Then, 50 µL of 1× MTT reagent was added to each well and the plate was kept at 37 °C for 4 h. The media was then carefully removed, and 100 µL of DMSO was added to each well followed by shaking

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for 10 min. The % viability was calculated as a ratio of absorbance at 490 nm of untreated and treated cells. Fluorescence imaging and Co-localization assay. Cells (~5 x 104) were plated onto glass bottom dish (In Vitro Scientific, D35-20-1-N) and allowed growing overnight. The cells were then incubated with probe 1, 3 or 4 (40 µM) in FBS free DMEM at 37 °C for 6 h. After that, the medium was removed, and the cells were washed with PBS (1×) three times. Epifluorescence images of the cells were then captured on an Olympus IX73 fluorescent inverted microscope with a 350 nm excitation filter and a 530 nm emission filter. For co-localization studies, MDA-MB-231 cells were incubated with 40 µM probe 1, 3 or 4 for 6 h. The cells were washed with PBS (1×) three times, and further incubated with 1 µM Lysotracker Red DND-99 at 37 °C for 20 min. After that, the medium was removed, and the cells were washed with cold PBS three times. Fluorescence imaging was performed under a Leica TCS SP5 confocal laser scanning microscope. Emission from amino luciferin was collected at green channel from 500 - 550 nm wavelength with the excitation at 405 nm, and emission from Lysotracker Red DND-99 was collected at red channel from 590 to 610 nm with excitation at 543 nm. Detection of CTB in MDA-MB-231 Cell lysates. MDA-MB-231 cells were seeded onto cell culture dishes (100×20 mm, corning), and allowed growing to ~ 90% confluence. The medium was removed and the cells were rinsed with cold PBS (1×) twice. Cold RIPA buffer (0.6 ml, sigma) were then added and incubated on an ice bath for five minutes. Then rapidly scrape the plate with a cell scraper to remove and lyse

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residual cells. Transferred the cell lysates to a 1.5 mL tube and the lysates were sonicated 30 s at ice bath. After centrifugation at 8,000 × g at 4 °C for 15 min, the supernatant was collected and the pH was carefully adjusted to 5.5 using 1 M HCl. To evaluate CTB activity, 50 µL of the lysate solution was added to a 96-well black plate and 50 µL of assay buffer containing 10 µM Cbz-Arg-Arg-AMC or probe 1 was added. The reaction mixture was incubated at 37 °C and the corresponding fluorescence intensity was recorded every minute on a microplate reader. To check the CTB-dependent fluorescence turn-on in cell lysates, CTB inhibitor (CA-074-Me, 2.0 µg/mL) was added into the lysates before incubation with Cbz-Arg-Arg-AMC or probe 1. RESULTS AND DISCUSSION

Figure 1. General design of lysosome-targeting fluorogenic probe for CTB. (a) The

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chemical structure of probe 1 and the proposed chemical conversion in response to CTB. (b) Illustration of the mechanism of action of probe 1 for fluorescence imaging of lysosomal CTB in living cells. (c) Chemical structure of the lysosome untargeted but CTB cleavable control probes 3 and 4. Design of Lysosome Targeting Fluorogenic Probe for CTB. Figure 1a shows the molecular structure of probe 1, consisting of a CTB recognition peptide substrate Cbz-Lys-Lys, a self-immolative linker PABA, a quenched amino luciferin fluorophore and a lysosome-targeting group morpholine. The dipeptide of Cbz-Lys-Lys has been demonstrated as a highly specific substrate for CTB when conjugating to the self-destructive linker PABA to design a prodrug-inspired fluorogenic probe.30 The application of morpholine as lysosome targeting group has been well explored to design molecular probes capable of accumulating in lysosomes.33-35 The fluorophore amino luciferin was chosen because of its high biocompatibility and good quantum yield in the visible light region upon activation.36,37 Figure 1b illustrates the proposed mechanism of action by which probe 1 reports intracellular CTB activity in living cells. Upon cell uptake, the fluorescence of probe 1 is silent in cytosol where the CTB activity is low. Then, the lysosome-targeting group morpholine could efficiently trigger the probe localized in lysosomes, where the active CTB could hydrolyze the Cbz-Lys-Lys dipeptide and thus trigger the spontaneous elimination of the self-immolative linker to remove the caging group, liberating probe 2 with strong green fluorescence. Finally, the protonation of morpholine in probe 2 under the acidic lysosomal environment (pH 4.5-6.0) to form the cationic charged product could help

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reduce its diffusion across lysosomal membrane and thus trapped inside lysosomes. As a result, strong green fluorescence distributing mainly in lysosomes could be observed, which correlates to the location and activity of CTB in living tumor cells. Two different control probes (3 and 4) containing the same CTB cleavable peptide substrate but lacking morpholine moiety were also designed to examine the role of lysosomal targeting group in probe 1 for the detection of intracellular CTB (Figure 1c). Synthesis and Characterization of Probes in Vitro. Probes 1, 3 and 4 were prepared in good yields according the approach outlined in Scheme S1. To demonstrate the proposed fluorescent product from probe 1 upon interaction with CTB as well as the lysosome targeting role of the morpholine unit, we also prepared the liberating product 2 (Scheme S2). The chemical structures of all these compounds were characterized by 1H NMR,

13

C NMR, ESI-MS and HMRS (Supporting

information). We firstly investigated the optical properties of the liberating fluorescent product 2, showing a strong fluorescence emission at 525 nm, a large Stock shift (∆λ = 171 nm) and a high quantum yield (0.21) in aqueous solution (Figure S1). The subsequent confocal study showed that product 2 can easily enter into live MDA-MB-231 cells, resulting in strong green fluorescence that colocalized well with red fluorescence from lysotracker red (DND-99) staining. In contrast, the incubation with the fluorophore of N-CBT that is lack of morpholine showed diffused fluorescence in the cytosol of cells. These results confirmed that the fluorescent product 2 had a high

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capacity to accumulate and stain the lysosomes of tumor cells with the help of morpholine group. Fluorescence Response toward CTB. The response of probe 1 to CTB was firstly examined by measuring the fluorescence spectra of 1 upon treatment with recombinant human CTB in enzyme reaction buffer (50 mM MES, 1 mM EDTA, 1 mM TCEP, 0.1% Triton X-100, pH 5.5, 37 °C). As shown in Figure 2a, probe 1 initially displayed weak fluorescence (λem = 450 nm). After incubation with CTB at 37 °C for 30 min, the fluorescence intensity of 1 (10 µM) in solution increased obviously, with the emission peak red shifting to 525 nm, which was the same as that of the fluorescent probe 2 (Figure S1b). The fluorescence turn-on ratio of probe 1 induced by CTB at 525 nm was calculated to be ~73-fold, with an easy-to-discern green color image observed under UV exposure at 365 nm (Figure 2a, insert). CTB-triggered proteolysis of probe 1 was then monitored in solution using high performance liquid chromatography (HPLC) analysis. As shown in Figure 2b, on incubation with CTB (1 µg/mL), probe 1 (10 µM, HPLC retention time, TR = 15.2 min) was nearly completely converted into product 2 (TR = 12.8 min) after 30 min, which was also confirmed by electrospray ionization mass spectroscopic (ESI-MS) analysis (Figure S2). Similar results were also observed for both control probes (3 and 4) containing the same CTB cleavable peptide substrate Cbz-Lys-Lys but lacking lysosome targeting group morpholine (Figure S3 and S4). These results demonstrate that CTB can efficiently cleave the peptide substrate in these probes in solution, releasing uncaged products with significant enhancement in fluorescence.

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The influence of pH to the CTB-mediated proteolysis of probe 1 was then investigated by incubating it (10 µM) with CTB (0.2 µg/mL) under different pH, and the fluorescence intensity at 525 nm was recorded as a function of time using a microplate reader. As seen in Figure 2c, CTB could readily cleave probe 1, resulting in a rapid increase in fluorescence intensity when conducting in acidic assay buffer (pH = 5.0-5.5). When the pH in the enzyme reaction solution increased to 6.0, the fluorescence intensity increased much slower and reached a plateau within 15 min. The maximum fluorescence intensity upon incubation with CTB was only ~30% in relative to that observed under more acidic conditions (pH = 5.0-5.5, Figure S5). There was nearly no change in fluorescence intensity observed when conducting within the pH range of 6.5-7.4. These results demonstrated that the optimum pH value for the reaction of probe 1 with CTB was within 5-6. As the lysosomal environment is well-known to be acidic (pH 4.5-6.0),38,39 the CTB-mediated fluorescence turn-on under acidic environment suggested that probe 1 was unique to detect the lysosomal CTB.

Figure 2. Analysis of probe 1 in response to CTB in vitro. (a) Fluorescence spectra of probe 1 (10 µM) before and after incubation with human recombinant CTB (1 µg/mL) at 37 °C for 30 min in CTB assay buffer (λex = 350 nm). Insert: Images of probe 1

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solutions before (-) and after (+) incubation with CTB under a UV lamp at 365 nm. (b) HPLC analysis of probe 1 (10 µM) before (black) and after (red) incubation with CTB (1 µg/mL) at 37 °C for 30 min. (c) Time-dependent fluorescence intensity changes of probe 1 (10 µM) incubated with 0.2 µg/mL CTB under different pH (λex = 350 nm, λem = 525 nm). Sensitivity, Selectivity and Enzymatic Kinetic Evaluation for CTB. Having demonstrated the response of probe 1 toward CTB, efforts were then made to investigate its sensitivity toward CTB using the optimized enzyme reaction conditions. We measured the fluorescence intensity of probe 1 (10 µM) at 525 nm following incubation with varying concentration of CTB at pH 5.5. As shown in Figure 3a, in the absence of CTB, probe 1 was stable in the enzyme reaction buffer, with little change in fluorescence intensity over the course of measurement. On incubation with CTB, it was clear to see that an increase in CTB concentration led to enhancements both

in

fluorescence

turn-on

rate

and

intensity,

indicating

a

CTB

concentration-dependent cleavage of probe 1. The fluorescence intensity at 525 nm was linearly proportional to the concentration of CTB from 0.02 to 0.5 µg/mL, with a detection limit of ~0.4 ng/mL (~0.01 nM, signal-to-noise, S/N = 3) (Figure 3b) obtained in less than 5 min. The sensitivity was comparable to that of other reported substrate-based fluorescence probes, but much higher than that of colorimetric probes.5 Interestingly, though probe 3 also showed a CTB concentration-dependent activation manner, there was a much slower activation rate observed compared to that of probe 1 under the same conditions (Figure S6a).

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The selectivity for CTB over other cathepsins was examined by comparing the fluorescence of probe 1 (10 µM) following incubation with 1 µg/mL CTB, CTL, CTS, CTD and CTB together with its inhibitor (CA-074-Me, for chemical structure, see Figure S12), respectively. As shown in Figures 3c and d, only CTB could produce a time-dependent enhancement in fluorescence intensity at 525 nm, with a significant ~50-fold turn-on ratio after 40 min. This enhancement was completely inhibited by the CBT inhibitor. Neither CTL, CTS nor CTD could activate probe 1 and give rise to enhanced fluorescence, suggesting that probe 1 was a specific substrate for CTB. This high selectivity was also observed for probe 3 containing the same CTB recognitive peptide sequence (Figures S6c and d).

Figure 3. (a) Time dependent fluorescence changes of probe 1 (10 µM) under different concentrations of CTB. (λex = 350 nm, λem = 525 nm). (b) The liner

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relationship between fluorescence intensities and enzyme concentrations. (c) Time-dependent fluorescence intensity changes of probe 1 (10 µM) alone, or probe 1 incubated with 1 µg/mL CTB, CTL, CTS, CTD, or CTB together with 2.0 µg/mL CTB inhibitor CA-074-Me (λex = 350 nm, λem = 525 nm). (d) Enhanced fluorescence intensity of probe 1 (10 µM) following incubation with 1 µg/mL CTB for 40 min. F/F0 represents the turn-on ratio of fluorescence intensity by CTB. Error bars are standard deviation (n = 3).

To further demonstrate the efficiency of probe 1 for CTB detection, its kinetic parameters, including the catalytic constant kcat and the Michaelis constant KM, were established from Lineweaver-Burk plots using the recombinant CTB (Figure S7). For comparison, the kinetic parameters of probe 3 and a commercialized CTB substrate Cbz-Arg-Arg-AMC were also measured and summarized in Table 1. Consistent with the above results, probe 1 showed high kinetic efficiency for CTB, with the kcat/KM value of approximately 154.6 mM-1 s-1. This value was comparable to that of Cbz-Arg-Arg-AMC (kcat/KM = 237.7 ± 19.0 mM-1 s-1), suggesting a reasonable cleavage efficiency of both probes toward CTB. In contrast, though probe 3 had a slight lower apparent KM value, it was significantly offset by a lower kcat, resulting in a much lower kinetic efficiency (kcat/KM = 52.5 ± 11.7 mM-1 s-1) when comparing to probe 1. These results suggested that the introduction of morphorline to probe 1 could not only promote the lysosomal delivery of the probe, but also contribute to improve kinetic efficiency for CTB detection, both of which were important for the efficient

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imaging of lysosomal CTB activity in cells. Table 1. Kinetics parameters of Cbz-Arg-Arg-AMC, probe 1 and probe 3a

a

Probe

KM (mM)

kcat (s-1)

kcat/KM (mM-1 s-1)

Cbz-Arg-Arg-AMC

0.11 ± 0.01

26.02 ± 0.42

237.7 ± 19.0

Probe 1

0.13 ± 0.01

21.90 ± 2.30

154.6 ± 11.8

Probe 3

0.09 ± 0.01

4.46 ± 0.72

52.5 ± 11.7

Kinetic data were measured in CTB assay buffer (50 mM MES, 1 mM EDTA, 1 mM

TCEP, 0.1% Triton X-100, pH 5.5) at 37 °C. All values indicated averages of three replicate experiments, and were shown as mean ± standard deviation. Imaging of Lysosomal CTB in Cells. To substantiate the possibility of probe 1 for imaging of CTB activities in cells, we firstly evaluated its cytotoxicity against both MDA-MB-231

and

HeLa

cancer

cells

using

the

3-[4,5-dimethyldhiazole-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay (Figure S8). After incubation with varying concentration of probe 1 or probe 3 (0, 10, 20, 40, 60, 80 µM) for 24 h, no adverse effect on cell viability was observed for either probe in both cell lines, demonstrating a high biocompatibility to live cells. The cell study conditions were then optimized by comparing the fluorescence intensity in MDA-MB-231 cells upon incubation with varying concentration of probe 1 (0, 10, 20, 40, 60, 80 µM) for different time (0, 1, 2, 4, 6, 8 h). The fluorescence images taken by epifluorescence microscopy showed both concentration- and incubation time-dependent cell uptake and fluorescence enhancement in living

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MDA-MB-231 cells, with a significant higher fluorescence intensity observed after incubation with 40 µM of probe 1 for 6 h than that of blank cells (Figure S9). Further increasing incubation concentration or prolonging incubation time did not translate into significant higher intracellular fluorescence intensities. Therefore, we selected the concentration of 40 µM and the incubation time of 6 h as the optimum conditions for next fluorescence imaging of lysosomal CTB with probe 1. MDA-MB-231 cells were incubated with 40 µM of probe 1 for 6 h, and the resulting fluorescence images were acquired on both epifluorescence (Figure S10) and confocal microscopes (Figure 4). To further demonstrate whether the enhanced fluorescence was indeed ascribed to the lysosomal CTB, the co-localization studies with the lysosomal tracker (Red DND-99) which could specifically label the lysosomes of MDA-MB-231 cells were also performed. As shown in Figure 4a, MDA-MB-231 cells incubated with probe 1 showed punctate bright-green intracellular fluorescence, which overlaid very well with the red fluorescence emitted from the lysosomal tracker, demonstrating that the enhanced green fluorescence was mainly distributed in the lysosomes of MDA-MB-231 cells. In contrast, MDA-MB-231 cells incubated with either the lysosome untargeted control probe (3 or 4) under the same conditions gave much weaker fluorescence (Figure 4b, Figure S10 and S11) as compared to that of probe 1. This was probably because that, the lack of morpholine group in both probes made them not efficiently delivered into lysosomes in MDA-MB-231 cells, thus preventing lysosomal CTB to cleave the peptide and promote efficient fluorescence turn-on. The CTB-induced fluorescence enhancement

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of probe 1 in cells was also confirmed by cell lysate studies, showing a higher fluorescence intensity in MDA-MB-231 cell lysate solution upon incubation with it, which was inhibited by the CTB inhibitor (CA-074-Me) (Figure S12). These results indicated that probe 1 is more preferential for fluorescence imaging of lysosomal CTB in cells.

Figure 4. Co-localization studies of MDA-MB-231 cells following incubation with probe 1 (or probe 3) and the Lysotracker Red. (a) Confocal fluorescence images of MDA-MB-231 cells after 6 h incubation with probe 1 showed co-localization of strong green fluorescence from the amino luciferin with red fluorescence from the Lysotracker Red. (b) Confocal fluorescence images of MDA-MB-231 cells after 6 h incubation with probe 3 showed weak green fluorescence with little co-localization with red fluorescence of the Lysotracker Red. Scale bar: 50 µm.

Having demonstrated the ability of probe 1 for imaging of intracellular CTB, we then applied it to differentiate cancer cells and normal tissue cells based on the fluorescence imaging studies. As shown in Figure 5, both HeLa and KB cancer cells that were found to overexpress CTB showed bright-green intracellular fluorescence

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after incubation with probe 1, which were similar to that observed in MDA-MB-231 cells (Figure S10). The incubation of human glioblastoma U87 cells with probe 1 under the same conditions resulted in slightly weaker intracellular fluorescence, implying lower CTB activity in U87 cells when comparing to HeLa and KB cancer cells. In contrast, there was much weaker fluorescence in either mouse fibroblast 3T3 cells or human embryonic kidney HEK-293 cells, suggesting significant lower CTB levels expressed on both normal tissue cells. From these fluorescence imaging results, it was reasonably to conclude that probe 1 was possible to differentiate CTB overexpressed cancer cells from normal tissue cells based on the simple fluorescence imaging studies. However, it should be mentioned that this method was not applicable to accurate quantification of CTB activity in these cells due to the lack of intracellular concentration of probe 1 as well as the dynamic intracellular environment.

Figure 5. Fluorescence imaging of CTB activity in HeLa, KB, U87 tumor cells and 3T3, HEK-293 normal cells. Cells were incubated with probe 1 (40 µM) at 37 °C for 6 h, and the epifluorescence images were acquired after removal of the incubation medium containing probe 1. Scale bar: 40 µm. CONCLUSION

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In summary, we have developed a CTB-activatable and lysosome-targeting fluorogenic probe 1 for fluorescence imaging of lysosomal CTB activity in living cancer cells. We have found that probe 1 could be selectively activated by CTB under the acidic lysosomal environment (pH 4.5-6.0), exhibiting high sensitivity and specificity for CTB detection. Fluorescence imaging results showed that probe 1 could efficiently enter and accumulate in the lysosomes of cancer cells, resulting in strong lysosomal fluorescence upon activation by CTB. The enhanced intracellular fluorescence was further applied as an indicator to report on lysosomal CTB activity in different cancer cells and normal tissue cells. The results demonstrated here clearly revealed that the introduction of morpholine could not only promote the lysosomal delivery of probe 1, but also improve its kinetic efficiency for CTB, both of which contributed greatly to efficient imaging of lysosomal CTB activity in cancer cells. ASSOCIATED CONTENT

Supporting Information. Supplementary Figures, schemes, synthetic procedures and structure characterization. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

*E-mail: [email protected]

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Notes The authors declare no competing financial interest. ACKNOWLEDGEMENT Financial supports from the National Natural Science Foundation of China (21505070, 21327902, 21372115 and 21632008) and Natural Science Foundation of Jiangsu Province (BK20150567) were acknowledged. REFERENCES (1) Baruch, A.; Jeffery, D. A.; Bogyo, M. Trends Cell Biol. 2004, 14, 29-35. (2) Zhang, W.; Gao.C. Sci. Bull. 2015, 60, 1973-1979. (3) Thomas, J. A. Chem. Soc. Rev. 2015, 44, 4494-4500. (4) Razgulin, A.; Ma, N.; Rao, J. Chem. Soc. Rev. 2011, 40, 4186-4216. (5) Kim, C. J.; Lee, D. I.; Kim, C.; Lee, K.; Lee, C. H.; Ahn, I. S. Anal. Chem. 2014, 86, 3825-3833. (6) Lee, H. W.; Heo, C. H.; Sen, D.; Byun, H.-O.; Kwak, I. H.; Yoon, G.; Kim, H. M. Anal. Chem. 2014, 86, 10001-10005. (7) Asanuma, D.; Sakabe, M.; Kamiya, M.; Yamamoto, K.; Hiratake, J.; Ogawa, M.; Kosaka, N.; Choyke, P. L.; Nagano, T.; Kobayashi, H.; Urano, Y. Nat. Commun. 2015, 6, 6463. (8) Gu, K.; Xu, Y.; Li, H.; Guo, Z.; Zhu, S.; Zhu, S.; Shi, P.; James, T. D.; Tian, H.; Zhu, W.-H. J. Am. Chem. Soc. 2016, 138, 5334-5340. (9) Zhu, L.; Ma, Y.; Kiesewetter, D. O.; Wang, Y.; Lang, L.; Lee, S.; Niu, G.;

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