Ratiometric Two-Photon Fluorescent Probe for Quantitative

Letter pubs.acs.org/ac

Ratiometric Two-Photon Fluorescent Probe for Quantitative Detection of β‑Galactosidase Activity in Senescent Cells Hyo Won Lee,†,‡ Cheol Ho Heo,†,‡ Debabrata Sen,† Hae-Ok Byun,§ In Hae Kwak,§ Gyesoon Yoon,*,§ and Hwan Myung Kim*,† †

Department of Energy Systems Research, Ajou University, Suwon, Gyeonggi-do 443-749, Korea Department of Biochemistry and Department of Biomedical Science, Ajou University School of Medicine, Suwon, Gyeonggi-do 443-721, Korea

§

S Supporting Information *

ABSTRACT: We reported a ratiometric two-photon fluorescent probe (SG1) for βgalactosidase (β-gal) and its application to quantitative detection of β-gal activity during cellular senescence in live cells and in aged tissues. This probe is characterized by a significant two-photon excited fluorescence, a marked blue-to-yellow emission color change in response to β-gal, easy loading, insensitivity to pH and reactive oxygen species (ROS), high photostability, and low cytotoxicity. In addition, we show that SG1 labeling is an effective tool for quantitative detection of senescence-associated βgal activity at the subcellular level in situ. This finding demonstrates that SG1 will find useful applications in biomedical research, including studies of cell aging.

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window, their short excitation wavelengths, and/or pH sensitivity, making the quantitative analysis of enzymatic activity difficult. An alternative approach for the detection of enzyme activity in live specimens is the use of two-photon microscopy (TPM) with an emission ratiometric probe. TPM, which employs two near-infrared photons as the excitation source, has become a powerful tool for imaging studies due to its advantages including localization of excitation, low photodamage, longer observation times, and greater tissue penetration depth.17−25 The ratiometric probe is suitable for quantitative analysis because the intensity ratio of hydrolyzed versus unreacted probe can eliminate the experimental effects such as probe distribution and incident laser power.21,22 Hence, there is a strong need to develop a ratiometric two-photon (TP) probe for β-gal. In this work, we report a ratiometric TP probe for β-gal (SG1, Scheme 1) and its application to quantitative analysis of β-gal activity during cellular senescence. This probe is composed of 6-(benzo[d]thiazol-2′-yl)-2-(methylamino)naphthalene as the TP fluorophore,21 β-D-galactopyranoside derived benzyl carbamate as the β-gal hydrolytic site,11 and 2,5,8,11-tetraoxatridecan-13-amine as the solubilizing group (Scheme 1).26 We anticipated that β-gal-mediated hydrolysis would cleave the carbamate linkage and liberate the amino

ell senescence is a process by which cells enter a permanent state of cell cycle arrest with diverse features, such as enlarged cell morphology, increase in intracellular reactive oxygen species (ROS), and loss of responsiveness to growth factors.1,2 With abundant evidence of senescent cells in multiple aged tissues, senescence is causally implicated in biological aging.3−5 Human β-galactosidase (β-gal) is a lysosomal exoglycosidase that removes galactose residues from various substrates, such as gangliosides, glycoproteins, sphingolipids, and keratin sulfate.6 Interestingly, abnormally accumulated β-gal activity has long been reported in senescent cells, allowing this senescence-associated β-gal (SA-β-gal) to be an important biomarker for senescence.7 However, the underlying mechanisms in the acquisition of SA-β-gal activity and its role in senescence and aging are still unknown. To address this in detail, it is crucial to monitor β-gal activity in living specimens at the subcellular level. The most common method to visualize SA-β-gal is a cytochemical assay using a synthetic β-gal substrate, X-gal (5bromo-4-chloro-3-indolyl-β-D-galactopyranoside), which yields a blue dimerized chromophore after enzymatic hydrolysis.3−5,8 The major drawback of X-gal is that cells must be fixed and manually counted.8−10 A second method is the use of FDG (fluorescein di-β-D-galactopyranoside) as a fluorescent probe for β-gal, which shows a fluorescence turn-on response after hydrolysis.8,11,12 This method suffers from low cell loading ability of FDG and its slow response rate. Recently, fluorescent turn-on probes for monitoring β-gal activity in live cells and in vivo have been developed.13−16 However, these probes are limited by having a turn-on response within a single detection © 2014 American Chemical Society

Received: August 19, 2014 Accepted: October 8, 2014 Published: October 8, 2014 10001

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Scheme 1. Structures of SG1 and 1

group as a strong electron donor, thereby increasing the internal charge transfer (ICT) character and shifting the emission maximum to the red region.27 The preparation of SG1 is described in the Supporting Information. The solubilities of SG1 and 1 in PBS buffer (10 mM, pH 7.4) were approximately 3−4 μM (Figure S1, Supporting Information), which is sufficient to stain the cells. Under this condition, SG1 and 1 exhibited absorption maxima (λabs) at 334 nm (ε = 2.70 × 104 M−1cm−1) and 378 nm (ε = 2.50 × 104 M−1cm−1), with emission maxima (λfl) at 461 nm (Φ = 1.00) and 540 nm (Φ = 0.16), respectively (Figure S2 and Table S1, Supporting Information). The larger Stokes shift observed in 1 compared to SG1 (162 vs 127 nm) can be attributed to the greater stabilization of the charge-transfer excited state in 1 that contains a stronger electron-donating group.27 The reaction of SG1 with β-gal produced 1 as the only product as confirmed by HPLC analysis (Figure S3, Supporting Information) and emission spectra (Figure 1a). The emission spectra of a 1 μM solution of SG1 treated with β-gal in PBS buffer (10 mM, pH 7.4, 37 °C) increased gradually at 540 nm with a concomitant decrease at 460 nm (Figure 1a). The ratio of the emission intensities (Fyellow/Fblue) at 410−460 nm (Fblue) and 520−570 nm (Fyellow) increased by 120-fold upon reaction with β-gal. This increase was suppressed in the presence of Dgalactose, a well-known competitive inhibitor of β-gal,28,29 in a dose-dependent manner, but not with D-glucose (Figure S4, Supporting Information), indicating that the ratiometric enhancement of SG1 is specific to β-gal activity. Further, the plot of the Fyellow/Fblue against the β-gal concentration ranging from 0 to 2.0 nM showed a linear relationship (Figure S5, Supporting Information), indicating that SG1 can detect β-gal at concentrations as low as 0.25 nM. Moreover, the response of this probe was much faster than that of commercial FDG (Figure 1b). The Michaelis−Menten constant (Km) of SG1 for the β-gal-catalyzed reaction was determined to be 1.73 ± 0.15 μM (kcat = 7.56 ± 0.17 s−1, Vmax = 16.3 ± 0.3 nmol mg−1 s−1) (Figure S6 and Table S2, Supporting Information). The Km value of SG1 was much lower than that for FDG (Km = 10.2 ± 1.2 μM, Table S2, Supporting Information), demonstrating that SG1 is more sensitive to β-gal activity than FDG. The TP action cross section (Φδmax) values of SG1 and 1 in PBS buffer were determined to be 21 and 58 GM at 740 and 750 nm, respectively (Table S1 and Figure S9, Supporting Information). The larger value for 1 can be attributed to the enhanced ICT between the donor and acceptor. These values can afford bright TPM images, as was observed (see below). Further, SG1 and 1 were pH insensitive at a biologically

Figure 1. Enzymatic reaction of SG1 and FDG with β-galactosidase. (a) Fluorescence response of SG1 (1 μM) with time (blue, initial spectrum; red, spectrum at completion) upon addition of 1 unit of βgalactosidase in PBS buffer (10 mM, pH 7.4, 37 °C). (b) Comparison of the time course of the fluorescence intensity between 1 μM SG1 (red) and 1 μM FDG (black) in PBS buffer (10 mM, pH 7.4, 37 °C) after addition of 1 unit of β-galactosidase. (Inset) Expansions of the initial fluorescence increase. The excitation wavelength was 376 nm for SG1 and 490 nm for FDG.

relevant pH range (Figure S7, Supporting Information). Moreover, SG1 exhibited no response upon addition of 200 μM concentration of various ROS (Figure S8, Supporting Information). Therefore, SG1 can serve as a ratiometric TP probe for β-gal activity with high sensitivity and with minimum interference from the pH and ROS. We then tested the utility of SG1 in detecting β-gal in the cellular environment. Primary cultured human diploid fibroblasts (HDFs) were incubated with 2 μM SG1 without any other complicated loading techniques.30−32 We obtained bright TPM images, presumably because of easy loading into the living cells and significant Φδmax values. Further, SG1 showed high photostability and no cytotoxicity as evidenced by cell viability tests using the MTS assay (Figures S12 and S13, Supporting Information). Next, we examined the potential applicability of SG1 to monitor the increases in endogenous SA-β-gal activity by employing a representative cell senescence model, replicative senescence. To establish this senescence, primary HDFs were continuously subcultured until they lost their cell division capacity as previously reported.33 During this process, doubling times (DT, time duration for doubling of cell population on a culture plate) and population doubling numbers (PD, the 10002

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Figure 2. (a) Pseudocolored ratiometric TPM images of HDFs incubated with 2 μM SG1 for 30 min at various stages of replicative senescence (from 2 to 20 days). Scale bars = 65 μm. Cells shown are representative images from replicate experiments (n = 6). (b) (filled bars) Average Fyellow/ Fblue intensity ratios in the TPM images. Images were acquired using 750 nm excitation with 30× magnification and fluorescent emission windows of 410−460 nm (Fblue) and 520−570 nm (Fyellow). (empty bars) Average percentages of SA-β-gal positive cells, subjected to X-gal cytostaining. Cells were fixed and loaded with X-gal at pH 6.0 for 12 h, and the number of blue colored cells were counted in the total population. Senescence displayed a progressive increase in cell size, reaching almost 10 times at 20 days. DT, doubling time; PD, population doubling number.

number of passages of the cell cultures obtained by sequential population doubling) were recorded (Table S3, Supporting Information). Of these processes, cellular DT began to increase progressively with enlarged cellular morphology after PD = 52, while it remained at about 2 days until their PD became 52 (Table S3, Supporting Information, and x-axis in Figure 2b). This result might indicate that a subset of the cell population begins to lose its capacity for cell division after PD reached 52. The TPM images of SG1-labeled HDFs were obtained and analyzed at various stages of this senescence process (Figure 2a). Upon TP excitation at 750 nm, the average emission ratios (Fyellow/Fblue) of young HDFs (DT2 and PD26) labeled with SG1 and 1 were 0.30 and 1.21, respectively (Figure S11, Supporting Information). The SG1 ratio increased gradually from 0.30 to 0.47 at DT5 (PD58) (Figure 2b). Thereafter, it began to rise dramatically from DT5 and eventually reached a value of 1.13 at DT20 (PD76). These results corresponded well with the results obtained by the X-gal staining (Figures 2b and S10, Supporting Information). Notably, SG1 labeling could sensitively detect the minor increases in SA-β-gal during the middle stages of senescence, demonstrating the capability of SG1 to delineate the acquisition profile of SA-β-gal during cell senescence. The ratiometric images of old HDFs (DT > 12) in Figure 2a clearly displayed intense spots (red color). It was previously reported that β-gal was highly expressed and accumulated in lysosomes in senescent cells.34,35 To assess whether the intense spots are indeed linked with the lysosome, the younger (DT2) and older (DT20) cells were costained with SG1 and LysoTracker Red (LTR), a well-known one-photon fluorescent probe for lysosomes (Figure 3). In DT20 cells, the TPM image of SG1 at the Fyellow channel merged well with the OPM image of LTR but not with those of DT2 cells (Figure 3). The Pearson’s colocalization coefficients (A), the correlation of the distribution of fluorescence intensity between two channels, were calculated using LAS AF software.36 The A values of SG1 and LTR for DT20 and DT2 cells were 0.73 and 0.32, respectively. Because the strong fluorescence intensity of the Fyellow channel is largely attributed to hydrolyzed product (1), this outcome indicated that the SA-β-gal activity mainly occurred in the lysosomal compartments, which concurred with literature results.34,35

Figure 3. (a, d) TPM, (b, e) OPM, and (c, f) merged images of HDFs colabeled with (a, d) SG1 and (b, e) LysoTracker Red. The images were taken at (a−c) the younger (DT2) and (d−f) older (DT20) stages, respectively. The wavelengths for TP and OP excitation were 750 and 514 nm, respectively, and the corresponding emissions were collected at 520−570 nm (SG1) and 600−650 nm (LTR). Scale bars = 40 μm. Cells shown are representative images from replicate experiments (n = 6).

To further investigate the genuine specificity of SG1 for SAβ-gal in situ, we employed premature senescence of HDFs which was directly induced by H2O2. After treatment of 150 μM H2O2, we monitored intracellular ROS level by flow cytometric analysis using 2,7-dichlorofluorescin diacetate (DCFH-DA), a widely used ROS-specific fluorescence probe.32,33 The ROS levels increased in a biphasic manner, showing the first peak within 1 h and the second increase after 24 h (Figure 4). The first peak seemed to be due to intracellular uptake of exogenous H2O2 whereas the second is considered to be linked with endogenous ROS generation triggered by the H2O2 stress because the second peak was measured after cells were replenished by fresh medium without H2O2. In this ROSmediated intracellular environment, the ratio of TPM images of SG1-labeled HDFs did not change during the first ROS peak period and only began to increase after 12 h (Figures 4 and S15, Supporting Information). Similar results were observed with the X-gal staining (Figure S14, Supporting Information). Moreover, because the X-gal staining is known to be affected by 10003

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In this work, we have developed SG1, a new emission ratiometric TP probe for β-gal that can quantitatively detect SA-β-gal activity in live HDFs and deep inside skin tissues. This probe shows a significant TP cross section, a marked blue-toyellow emission color change in response to β-gal, easy loading into cells, insensitivity to pH in the physiological range and to ROS, high photostability, and low cytotoxicity. In addition, ratiometric TPM imaging using SG1 is an effective tool for monitoring different β-gal activities at the subcellular level in situ. This probe may find useful applications in biomedical research, including studies of cell aging.

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ASSOCIATED CONTENT

* Supporting Information S

Figure 4. (filled bars) Average Fyellow/Fblue intensity ratios in the TPM images of primary young HDFs labeled with 2 μM SG1. The cells were pretreated with 150 μM H2O2 for 0, 1, 3, 6, 12, 24, 48, and 72 h, respectively, and were incubated with probe, and the TPEF were collected using 750 nm excitation and fluorescent emission windows of 410−460 nm (Fblue) and 520−570 nm (Fyellow). (empty bars) Intracellular ROS levels of H2O2-treated HDFs monitored by flow cytometric analysis after staining the cells with 10 μM DCFH-DA.

Synthesis, photophysical, and imaging experiments; additional figures and images (Figures S1−S23, Tables S1−S3). This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +82-31-219-1615. *E-mail: [email protected].

buffer pH,9,10 we investigated the effect of various pH (4.0− 8.0) on the ratio of SG1-labeled DT2 and DT20 cells, compared with X-gal. When using X-gal, the senescent cells with enlarged cell morphology were more specifically detected in the old cell population only at pH 6.0 (Figure S16, Supporting Information), but that was not the case with lower pH values (6.0) cannot be applied. These results explain the conventional assay’s dependency on the narrow range of pH, as reported.9,10 On the other hand, the Fyellow/Fblue ratio of SG1-labeled cells displayed an unperturbed manner by pH ranging from 4.0 to 8.0 (Figure S17, Supporting Information). Consequently, SG1 is capable of monitoring β-gal activity, especially SA-β-gal, in live cells without interference from the intracellular ROS and pH. Finally, we tested the utility of SG1 in tissue imaging. The skin tissue slices were collected from 7- and 26-month-old Sprague−Dawley rats, respectively. The TPM images of SG1labeled tissues clearly showed the distribution of β-gal activity at a depth of about 140 μm (Figure 5a,b). Further, the average emission ratio in older tissue (26-month) increased to 0.78 from 0.55 measured in younger tissue (7-month) (Figure 5c). Hence, SG1 is clearly capable of detecting β-gal activity deep inside of tissues using ratiometric TPM imaging.

Author Contributions ‡

H.W.L. and C.H.H. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Research Foundation (NRF) grants funded by the Korean Government (2012R1A2A1A03670456 and 2012R1A5A2048183). We thank the Aging Tissue Bank of Pusan, Korea, for the supply of aged tissues.

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Figure 5. Pseudocolored ratiometric TPM images (Fyellow/Fblue) of (a) 7-month-old and (b) 26-month-old Sprague−Dawley rat skin tissues stained with 10 μM SG1. Images (30× magnification) were acquired at a depth of 140 μm using 750 nm excitation and fluorescent emission windows of 410−460 nm (Fblue) and 520−570 nm (Fyellow). Scale bars = 65 μm. (c) The average emission ratio in older (26-month) and younger (7-month) tissue. 10004

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